Inactivation of the retinoblastoma gene in a human lung carcinoma cell line detected by single-strand conformation polymorphism analysis of the polymerase chain reaction product of cDNA.

Combined use of a simple, sensitive method of DNA analysis of nucleotide substitutions, namely, single-strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP), and the reverse transcriptase reaction (RT) is an effective method for mRNA analysis. We used this RT-PCR-SSCP method to detect abnormal retinoblastoma (RB) gene transcripts in human tumor cell lines. Results showed the presence of two types of RB gene transcripts in a giant cell lung carcinoma cell line Lu65: a minor mRNA species with a base substitution that created a stop codon in the nucleotide sequence corresponding to exon 2 of the gene, and a major species of mRNA without the nucleotide sequence corresponding to that of exon 2. PCR-SSCP analysis of the genomic DNA also revealed that Lu65 cells contained the mutated RB allele, but not the normal allele. These results suggested that in Lu65 cells, both RB alleles were inactivated. The transcript without the exon 2 sequence, probably due to alternative splicing, was also found in all the other human cells examined, as a very minor species.

Local and overall conformations of DNA double helices with the A - T base pairs.

Raman spectra have been observed of two different poly[d(A-T)].poly[d(A-T)] fibers, whose X-ray diffractions were confirmed to be purely of A and B forms. A number of spectral differences were found between the A and B forms of this DNA duplex, and they were ascribed to local conformational differences in the adenosine, thymidine and phosphodiester portions. The ascription was made on the basis of a separate series of Raman examinations on six crystals involving adenosine or thymidine, and fifteen other nucleotide crystals, whose structures are all known by previous crystallographic works. By taking these structure-spectrum correlations thus obtained into account, a Raman spectroscopic investigation was made of a few double-helical DNAs in aqueous solutions. It has been concluded that both poly[d(A-T)].poly[d(A-T)] and poly(dA).poly(dT) have a C2'endo-anti adenosine, C2'endo-anti thymidine, a b-type mainchain (beta = 160 +/- 15 degrees, gamma = 45 +/- 15 degrees, delta = 140 +/- 10 degrees) and an a2-type mainchain (beta = 210 +/- 10 degrees, gamma = 45 +/- 15 degrees, delta = 140 +/- 10 degrees) not only in low-salt medium but also in 6.6 M CsF solutions, where beta, gamma and delta are the torsion angles around O5'-C5', C5'-C4' and C4'-C3' axes, respectively. Poly(rA).poly(dT), on the other hand, was considered to have a heteronomous duplex structure, in which the poly(rA) strand has a C3'endo-anti adenosine and a1-type mainchain (beta = 175 +/- 25 degrees, gamma = 45 +/- 15 degrees, delta = 80 +/- 10 degrees) whereas the poly(dT) strand has a C2'endo-anti thymidine and b-type mainchain.

Structure, backbone dynamics and interactions with RNA of the C-terminal RNA-binding domain of a mouse neural RNA-binding protein, Musashi1.

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in the regulation of asymmetric cell division. Musashi1 contains two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), RBD1 and RBD2. Our previous studies showed that RBD1 alone binds to RNA, while the binding of RBD2 is not detected under the same conditions. Joining of RBD2 to RBD1, however, increases the affinity to greater than that of RBD1 alone, indicating that RBD2 contributes to RNA-binding. We have determined the three-dimensional solution structure of the C-terminal RBD (RBD2) of Musashi1 by NMR. It folds into a compact alpha beta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. Special structural features of RBD2 include a beta-bulge in beta2 and a shallow twist of the beta-sheet. The smaller 1H-15N nuclear Overhauser enhancement values for the residues of loop 3 between beta2 and beta3 suggest that this loop is flexible in the time-scale of nano- to picosecond order. The smaller 15N T2 values for the residues around the border between alpha2 and the following loop (loop 5) suggest this region undergoes conformational exchange in the milli- to microsecond time-scale. Chemical shift perturbation analysis indicated that RBD2 binds to an RNA oligomer obtained by in vitro selection under the conditions for NMR measurements, and thus the nature of the weak RNA-binding of RBD2 was successfully characterized by NMR, which is otherwise difficult to assess. Mainly the residues of the surface composed of the four-stranded beta-sheet, loops and C-terminal region are involved in the interaction. The appearance of side-chain NH proton resonances of arginine residues of loop 3 and imino proton resonances of RNA bases upon complex formation suggests the formation of intermolecular hydrogen bonds. The structural arrangement of the rings of the conserved aromatic residues of beta2 and beta3 is suitable for stacking interaction with RNA bases, known to be one of the major protein-RNA interactions, but a survey of the perturbation data suggested that the stacking interaction is not ideally achieved in the complex, which may be related to the weaker RNA-binding of RBD2.

Structure of the C-terminal RNA-binding domain of hnRNP D0 (AUF1), its interactions with RNA and DNA, and change in backbone dynamics upon complex formation with DNA.

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP) -type RNA-binding domains (RBDs), each of which can specifically bind to the UUAG-sequence. hnRNP D0 also binds specifically to single-stranded d(TTAGGG)(n), the human telomeric DNA repeat. We have already reported the structure and interactions with RNA of the N-terminal RBD (RBD1). Here, the structure of the C-terminal RBD (RBD2) determined by NMR is presented. It folds into a compact alpha beta structure comprising an antiparallel beta-sheet packed against two alpha-helices, which is characteristic of RNP-type RBDs. In addition to the four beta-strands commonly found in RNP-type RBDs, an extra beta-strand, termed beta 4(-), was found just before the fourth beta-strand, yielding a five-stranded beta-sheet. Candidate residues of RBD2 involved in the interactions with RNA were identified by chemical shift perturbation analysis. Perturbation was detected on the beta-sheet side, not on the opposite alpha-helix side, as observed for RBD1. It is notable that the beta 4(-) to beta 4 region of RBD2 is involved in the interactions in contrast to the case of RBD1. The chemical shift perturbation analysis also showed that RBD2 interacts with DNA in essentially the same way as with RNA. Changes in the backbone dynamics upon complex formation with DNA were examined by means of model free analysis of relaxation data. In free RBD2, the beta 4(-) to beta 4 region exhibits slow conformational exchange on the milli- to microsecond time scale. The exchange is quenched upon complex formation. The flexibility of free RBD2 may be utilized in the recognition process by allowing different conformational states to be accessed and facilitating induced fit. Additionally, faster flexibility on the nano- to picosecond time scale was observed for loop 3 located between beta 2 and beta 3 in free RBD2, which is retained by the complex as well.

Interaction of the C-terminal domain of the E. coli RNA polymerase alpha subunit with the UP element: recognizing the backbone structure in the minor groove surface.

The C-terminal domain of the alpha-subunit of Escherichia coli RNA polymerase (alphaCTD) is responsible for transcriptional activation through interaction with both activator proteins and UP element DNA. Previously, we determined the solution structure of alphaCTD. Here, we investigated the interaction between alphaCTD and UP element DNA by NMR. DNA titration curves and intermolecular NOE measurements indicate that alphaCTD can bind to multiple sites on the UP element DNA. Unlike many transcription factors, alphaCTD does not have a strict base sequence requirement for binding. There is a good correlation between the strength of the interaction and the extent of intrinsic bending of the DNA oligomer estimated from the gel retardation assay. We propose that alphaCTD recognizes the backbone structure of DNA oligomers responsible for the intrinsic bending. Moreover, NMR studies and drug competition experiments indicated that alphaCTD interacts with the UP element on the minor groove side of the DNA. The C-terminal end of helix-1, the N-terminal end of helix-4, and the loop between helices 3 and 4 are used for the interaction. Based on these observations, we propose a model for the UP element-alphaCTD complex.

An intramolecular quadruplex of (GGA)(4) triplet repeat DNA with a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad, and its dimeric interaction.

The structure of d(GGAGGAGGAGGA) containing four tandem repeats of a GGA triplet sequence has been determined under physiological K(+) conditions. d(GGAGGAGGAGGA) folds into an intramolecular quadruplex composed of a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad. Four G-G segments of d(GGAGGAGGAGGA) are aligned parallel with each other due to six successive turns of the main chain at each of the GGA and GAGG segments. Two quadruplexes form a dimer stabilized through a stacking interaction between the heptads of the two quadruplexes. Comparison of the structure of d(GGAGGAGGAGGA) with the reported structure of d(GGAGGAN) (N=G or T) containing two tandem repeats of the GGA triplet revealed that although the two structures resemble each other to some extent, the extension of the repeats of the GGA triplet leads to distinct structural differences: intramolecular quadruplex for 12-mer versus intermolecular quadruplex for 7-mer; heptad versus hexad in the quadruplex; and three sheared G:A base-pairs versus two sheared G:A base-pairs plus one A:A base-pair per quadruplex. It was also suggested that d(GGAGGAGGAGGA) forms a similar quadruplex under low salt concentration conditions. This is in contrast to the case of d(GGAGGAN) (N=G or T), which forms a duplex under low salt concentration conditions. On the basis of these results, the structure of naturally occurring GGA triplet repeat DNA is discussed.

Structure and interactions with RNA of the N-terminal UUAG-specific RNA-binding domain of hnRNP D0.

Heterogeneous nuclear ribonucleoprotein (hnRNP) D0 has two ribonucleoprotein (RNP)-type RNA-binding domains (RBDs), each of which can bind solely to the UUAG sequence specifically. The structure of the N-terminal RBD (RBD1) determined by NMR is presented here. It folds into a compact alphabeta structure comprising a four-stranded antiparallel beta-sheet packed against two alpha-helices, which is characteristic of the RNP-type RBDs. Special structural features of RBD1 include N-capping boxes for both alpha-helices, a beta-bulge in the second beta-strand, and an additional short antiparallel beta-sheet coupled with a beta-turn-like structure in a loop. Two hydrogen bonds which restrict the positions of loops were identified. Backbone resonance assignments for RBD1 complexed with r(UUAGGG) revealed that the overall folding is maintained in the complex. The candidate residues involved in the interactions with RNA were identified by chemical shift perturbation analysis. They are located in the central and peripheral regions of the RNA-binding surface composed of the four-stranded beta-sheet, loops, and the C-terminal region. It is suggested that non-specific interactions with RNA are performed by the residues in the central region of the RNA-binding surface, while specific interactions are performed by those in the peripheral regions. It was also found that RBD1 has the ability to inhibit the formation of the quadruplex structure.

Cytokine regulation of the rat proopiomelanocortin gene expression in AtT-20 cells.

Although cytokines are known to be involved in the regulation of ACTH secretion, their effects, along with the molecular mechanisms, on POMC gene expression are not thoroughly characterized. In this study we examined the effects of representative cytokines on transcription of the POMC gene in corticotrophs in vitro using AtT20PL, a clone of the AtT20 cell line stably transfected with approximately 0.7 kilobase of the rat POMC 5'-promoter-luciferase fusion gene. In each experiment, cells were incubated with the cytokine tested, and the changes in POMC 5'-promoter activity were determined by a luciferase assay. The results showed that interleukin-1beta (IL-1beta) stimulated promoter activity in a biphasic manner [weak short term effects (2-3 h) followed by potent long term effects (>12-16 h)]. Tumor necrosis factor-alpha had similar effects, but much less potency. IL-6 showed a profound stimulatory, but only a long term (>20 h), effect. IL-2 did not influence POMC expression. In contrast, interferon-alpha (IFN alpha) and IFN-gamma showed acute stimulatory effects (approximately 4 h) followed by marked inhibitory effects (>8 h). Although the acute effects of IL-1beta, IL-6, and tumor necrosis factor-alpha alone were minimal, they significantly potentiated the stimulatory effect of CRH on POMC expression. Finally, pretreatment of the cells with a broad spectrum tyrosine kinase inhibitor, genistein, abolished or significantly diminished the effects of all cytokines except IFNs. Our results suggest that 1) each cytokine tested has a distinct effect on POMC gene expression; 2) there are positive cross-talk effects between CRH and cytokines at the corticotroph level; and 3) tyrosine phosphorylation cascades are involved in the intracellular signaling mechanisms of some cytokines.

Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells. I: Effects of the common secretagogues.

Although the effects of the various secretagogues on corticotropin (ACTH) secretion have been well studied, their effects on the POMC gene expression have not been thoroughly characterized. In this study, we established a new model system using the AtT20 mouse corticotroph tumor cell line transfected stably with a plasmid containing 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene. The responsiveness to exogenous CRH improved markedly when the cells were cultured with low serum medium (1% FBS) compared with serum rich medium (10%). Using this culture condition, we examined the effects of not only CRH but also other secretagogues such as catecholamines, vasopressin, and angiotensin II, upon the transcriptional activity of the POMC gene. CRH stimulated POMC promoter activity (3.5-fold increase) as well as cAMP generation and ACTH secretion in a dose- and time-dependent manner, with the maximal effect being observed 3-5 h after the start of incubation. Catecholamines, especially epinephrine (10 nM and above), also stimulated all parameters, although less potently than CRH, and the effect was mimicked by the beta-, but not alpha-adrenergic, agonist, suggesting the involvement of the beta-adrenergic receptor. The combined effects of epinephrine and CRH were greater in all parameters than those of CRH alone, and the effects of both hormones were completely blocked by H89, an inhibitor of protein kinase A. Vasopressin and angiotensin II showed minimal effects on POMC expression. Our results suggest that 1) catecholamines, as well as CRH, positively regulate the POMC gene at physiological concentrations; 2) the cAMP-PKA system is the common intracellular signaling pathway for CRH and catecholamines; and 3) vasopressin and angiotensin II also have weak but significant stimulatory effects on POMC promoter activity.

Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells. II: Effects of the pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP), members of the glucagon-secretin family, have recently been suggested to be involved in the regulation of corticotropin (ACTH) secretion. In this study, we examined the effects of both peptides on POMC gene expression. Using AtT20PL, a clone of the AtT20 mouse corticotroph tumor cells stably transfected with 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene, the effects of both peptides on the POMC promoter activity were estimated by a luciferase assay. PACAP stimulated POMC 5' promoter activity as well as cAMP generation and ACTH secretion in a dose- and time-dependent manner, with the maximal effect being observed 3 h after the start of incubation. A similar effect was observed with VIP. Although the combined effects of PACAP/CRH or VIP/CRH were greater than that of either hormone alone, no such effect was observed between PACAP and VIP. Furthermore, RT-PCR analysis showed the presence of only the PVR3 receptor subtype in this cell line, which is known to have a similar affinity to PACAP and VIP, indicating that both peptides exert their effects through the same receptor. In contrast to the effect of CRH, which was completely abolished by a protein kinase A inhibitor H89, the effects of PACAP/VIP on POMC expression persisted during H89 treatment, suggesting the involvement of alternative intracellular signaling pathway(s) distinct from the protein kinase A system. Our results suggest that PACAP and VIP have positive effects on POMC gene expression and that multiple signaling pathways are involved in the transcriptional event.

Structural properties and RNA-binding activities of two RNA recognition motifs of a mouse neural RNA-binding protein, mouse-Musashi-1.

mouse-Musashi-1 (m-Msi-1) is an RNA-binding protein, abundantly expressed in the developing mammalian central nervous system (CNS). m-Msi-1 contains two RNA recognition motifs (RRMs). In this study, we found that the N-terminal RRM of m-Msi-1 (MMA) binds strongly to poly(G) and weakly to poly(U) in a way similar to that of the full-length m-Msi-1 protein characterized previously. The C-terminal RRM of m-Msi-1 (MMB), however, does not bind to RNA. In addition, the circular dichroism (CD) spectra of the two RRMs showed that the alpha-helical content of MMA is significantly higher than that of MMB, indicating that some differences in the secondary structure may be responsible for the distinct RNA binding properties of MMA and MMB.

An NMR study on the conformation of a deoxyoligonucleotide duplex, d(GGGGCCCC)2, and its complex with chromomycin.

The conformation of drug-free d(GGGGCCCC)2 and the chromomycin-d(GGGGCCCC)2 complex in aqueous solution were studied by NMR spectroscopy. The present study has indicated that free d(GGGGCCCC)2 takes the B form in solution, although it takes the A form in the crystalline state. The NMR spectrum of the complex indicated that chromomycin binds as a symmetry-related dimer to the minor groove of the central four residues of d(GGGGCCCC)2. The drastic conformational change in the central four residues of d(GGGGCCCC)2 on going from the B form family to the A form was demonstrated by the characteristic NOEs and coupling patterns. The change seems to be indispensable for accommodation of the bulky chromomycin dimer in the minor groove. On the basis of the intermolecular NOEs between chromomycin and d(GGGGCCCC)2, the structure of the complex has been constructed and refined by energy minimization.

Activation and repression of the activity of a lead ribozyme by the combination of Pb2+ and Mg2+.

The effect of Pb2+ and Mg2+ on the activity of a lead ribozyme with modified sequences has been studied. At low Pb2+ concentrations, cleavage at a previously reported site (site a) is observed. At higher Pb2+ concentrations, cleavage at a new site (site b) adjacent to site a is observed, while the cleavage at site a is repressed. On the addition of a certain amount of Mg2+, the cleavage at site a is enhanced by almost fourfold, while the cleavage at site b is repressed. Further addition of Mg2+ represses the cleavage at both sites. CD analysis indicates that the structure and stability of the lead ribozyme change depending on the metal conditions. Activation and repression of the activity by the combination of Pb2+ and Mg2+ are rationalized by considering that the two metals compete with each other for binding at two metal-binding sites.

Properties of a hammerhead ribozyme with deletion of stem II.

The properties of a mutant hammerhead ribozyme system, which consists of two RNA oligomer strands and in which stem II is deleted (replaced with a UUUU loop), are described. The effects of temperature, pH, and metal ions on the cleavage reaction were similar to those for the parent ribozyme with stem II. The mutant ribozyme showed a much lower cleavage rate (kcat = 0.04 min-1) in the presence of 10 mM MgCl2, where the parent ribozyme showed full cleavage activity. However, increasing the concentration of MgCl2 from 10 to 100 mM restored the cleavage activity of the mutant ribozyme to the original level (kcat = 0.2 min-1). CD titration experiments with MgCl2 using a noncleavable substrate were carried out. Deletion of stem II resulted in an about 20-fold reduction of the apparent Mg2+ binding affinity when the Mg2+ concentrations of half-saturation are compared. The results were analyzed by curve-fitting analysis and compared with those for the parent ribozyme. The analysis showed that the Mg2+ concentration dependence data in CD and cleavage experiments for the mutant enzyme can be explained by a two Mg2+ ion binding mechanism. These results that stem II is important for maintaining the conformation of the catalytic core suitable for Mg2+ binding.

Dimerization and DNA binding facilitate alpha-helix formation of Max in solution.

Max is a basic region/helix-loop-helix/leucine zipper (b/HLH/Z) protein that forms a hetero-complex with the Myc family proteins Myc, Mad, and Mxi1, and a homo-complex with itself. These complexes specifically bind to double-stranded DNA containing CACGTG sequences. Here, we report on the structural properties in aqueous solution of a 109-amino-acid protein, Max110, corresponding to the N-terminal domain of Max containing the b/HLH/Z motif (residues 2-110), as characterized by combined use of circular dichroism (CD) and sedimentation equilibrium experiments. The results showed that the alpha-helical content of Max110 increases with increasing protein concentration. The sedimentation equilibrium data indicated that Max110 exists as a monomer at low protein concentration, and forms a dimer at high protein concentration. Further increases in the alpha-helical content of Max110 occur upon addition of DNA with the CACGTG recognition sequence. Thus, dimerization and binding to DNA of Max both favor an increase of the alpha-helical content.

Properties of hepatitis delta virus ribozyme, which consists of three RNA oligomer strands.

Properties of a hepatitis delta virus (HDV) RNA ribozyme system, which consists of three RNA oligomer strands (substrate 8-mer; enzyme 16-mer plus 35-mer) and contains a hybrid sequence of genomic and antigenomic RNA cores, are reported. Effects of Mg2+ concentration, divalent metal ion species, pH, and temperature on the cleavage activity were examined. The substrate cleavage activity increased with increasing Mg2+ concentration (0-100 mM). Ca2+ and Mn2+ ions were the most effective divalent cations and Mg2+ was less effective. The cleavage activity increased with increasing pH (5-7.5). The optimum temperature for the cleavage activity was 25-40 degrees C. The Mg2+ concentration, pH and temperature dependencies are different from those reported for the single-strand ribozymes (about 90-mer) although the divalent metal ion preference is very similar. Conformational change induced by Mg2+ ion titration was monitored by CD. The CD data and the activity-Mg2+ concentration data were analyzed by curve-fitting analysis using equations derived for multiple metal ion binding mechanisms. The data can be explained by a model in which three Mg2+ ions bind to one ribozyme unit.

Hairpin structure of an RNA 28-mer, which contains a sequence of the enzyme component of a hammerhead ribozyme system: evidence for tandem G:A pairs that are not of side-by-side type.

An RNA 28-mer (Rz28) was obtained as a major product by in vitro transcription with T7 RNA polymerase of a promoter-template DNA, which contains a sequence for the enzyme component, RNA 24-mer (Rz24), of a mutant hammerhead ribozyme system. Sequence analysis and enzymatic probing study showed that Rz28 has 4 extra nucleotides at the 3'-terminus, the sequence of which is complementary to that of the 5'-terminal sequence of Rz24, and forms a stable hairpin structure. NMR studies using a 15N-guanine-labeled derivative suggested that Rz28 contains tandem G:A pairs that are not of the side-by-side type which is found in the crystal structure of hammerhead ribozyme complexes. Comparison of the HMQC spectra of 15N-guanine-labeled Rz28 and Rz24 suggested that Rz24 also contains the same type of tandem G:A pairs.

Analysis of tubulin isoforms by two-dimensional gel electrophoresis using SDS-polyacrylamide gel electrophoresis in the first dimension.

A two-dimensional electrophoretic system using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the first dimension and isoelectric focusing (IEF) in the second dimension was devised. In spite of its simplicity, this method could show a markedly high resolution for tubulin isoforms and moreover could classify them into alpha- or beta-tubulin as a two-dimensional profile. With this method, seven alpha- and four beta-tubulin isoforms could be detected within axoneme from Tetrahymena cilia. Moreover this method could also resolve tubulin isoforms from the rabbit brain. These results indicate that the present two-dimensional gel electrophoresis is a useful tool for the electrophoretic analysis of tubulin isoforms from various sources.

Evaluation of the induction of hepatic drug-metabolizing enzymes in the rat and mouse treated with temazepam using trimethadione as the indicator.

In the present paper we report the effect of high doses of temazepam on drug-metabolism induction in rat and mouse. Temazepam induced a dose-dependent and reversible increase of hepatic microsomal drug-metabolizing enzymes in the rat, dose-correlated increase of dimethadione/trimethadione ratios in plasma of the rat, and dose-related shortening of hexobarbital-induced sleeping time in the mouse. In this study trimethadione is considered a good indicator for estimating the state of hepatic enzyme induction.

[Clinicopathological study of oligodendroglioma with special reference to immunohistochemical investigation].

The present study was undertaken to evaluate the utility of pathologic features and specific immunohistochemical studies in estimating the prognosis of oligodendroglioma. The pathological diagnosis of an oligodendroglioma was made on HE stained-sections according to WHO classification. Sixteen oligodendrogliomas, twelve mixed oligoastrocytomas and ten anaplastic oligodendrogliomas were immunotested by the peroxidase-antiperoxidase (PAP) method with anti-GFAP serum, anti-S-100 serum and anti-MBP (Myelin basic protein) serum and by the avidin biotin peroxidase-complex (ABC) method with anti-vimentin serum and ant-Leu 7 monoclonal antibody. GFAP positive cells were interpreted as reactive astrocytes, neoplastic astrocytes and neoplastic oligodendrocytes, S-100 positive cells were interpreted as reactive astrocytes and neoplastic astrocytes. Leu 7 positive cells were found in only one case of anaplastic oligodendroglioma. Anti-Leu 7 could not be considered as a specific marker for oligodendroglioma. Of the anaplastic oligodendroglioma 60% displayed MBP positively and 70% displayed vimentin positively. NSE positive cells were found in a few anaplastic oligodendrogliomas. The present study has not so far uncovered any marker that is restricted to oligodendrogliomas. However GFAP may be useful to assess the extent of reactive astrocytes and neoplastic astrocytes in the oligodendroglioma or mixed oligoastrocytoma. MBP and vimentin will help to determine the malignancy of oligodendroglioma.