Analysis of the role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain phosphorylation through structure-function studies of TFIIE-alpha.

The general transcription factor TFIIE recruits TFIIH at a late stage of transcription initiation complex formation and markedly stimulates TFIIH-dependent phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II. To study this function of TFIIE in more detail, systematic deletion mutations were introduced into the large subunit of TFIIE (TFIIE-alpha) and were analyzed with regard to their effects on TFIIH-dependent CTD phosphorylation, TFIIE-dependent basal and enhancer-dependent transcription, and interactions of TFIIE-alpha with both TFIIE-beta and TFIIH. The amino (N)-terminal half of TFIIE-alpha, which possesses several putative structural motifs, was sufficient for the phosphorylation and transcription activities and for TFIIE-beta interactions, whereas a site effecting both strong interactions with TFIIH and large stimulatory effects on transcription and CTD phosphorylation was localized to an acidic region near the carboxy (C) terminus. The fact that these activities appear to be tightly linked supports the idea that TFIIE interacts physically and functionally with TFIIH and that CTD phosphorylation is essential for transcription under normal conditions. The present results suggest that TFIIE, via its effect on TFIIH, may act as a checkpoint for formation of a preinitiation complex.

Studies of nematode TFIIE function reveal a link between Ser-5 phosphorylation of RNA polymerase II and the transition from transcription initiation to elongation.

The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIEalpha and ceTFIIEbeta). ceTFIIEbeta could partially replace hTFIIEbeta, whereas ceTFIIEalpha could not replace hTFIIEalpha. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIEalpha did not bind tightly to hTFIIEbeta, and ceTFIIEbeta showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIEbeta induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIEbeta was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.

Mechanism of promoter melting by the xeroderma pigmentosum complementation group B helicase of transcription factor IIH revealed by protein-DNA photo-cross-linking.

The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of transcription factor IIH (TFIIH) is essential for promoter melting prior to transcription initiation by RNA polymerase II (RNAPII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions -9 to +2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions -5 and +1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed.

Participation of common surface receptor(s) in the activation of murine macrophages by Sarcophaga lectin and wheat germ agglutinin.

Mouse peritoneal macrophage surface proteins which bind Sarcophaga lectin were studied. Two major binding proteins with molecular masses of 170 and 110 kDa were identified. Sarcophaga lectin and wheat germ agglutinin were found to share common binding proteins for activating macrophages, although their hapten sugars are different. Antibody raised against the Sarcophaga lectin-binding proteins inhibited both the production of tumor-specific cytotoxic protein by the macrophage-like cell line J774.1 cells and the lectin-dependent macrophage-mediated cytotoxic reaction induced by Sarcophaga lectin or wheat germ agglutinin. Thus the 170-kDa and/or 110-kDa protein is important in activation of macrophages.

Identification of target proteins participating in a lectin-dependent macrophage-mediated cytotoxic reaction.

Membrane proteins of mouse macrophages and mammary tumor cells having affinity to Sarcophaga lectin were isolated by affinity chromatography. The electrophoretic profiles and antigenicities of lectin-binding proteins from macrophages and tumor cells were different. Antibody raised against tumor cell lectin-binding proteins inhibited both the binding of the lectin to tumor cells and the lectin-dependent macrophage-mediated cytotoxic reaction. However, it did not inhibit the binding of the lectin to macrophages. It was suggested that the same lectin molecule transmitted different stimuli to macrophages and tumor cells via different receptor proteins.

Comparison of binding proteins on the surface of murine tumor cells for two lectins active in the lectin-dependent macrophage-mediated cytotoxic reaction.

The binding proteins for Sarcophaga lectin and wheat germ agglutinin on the surface of Ehrlich ascites tumor cells were compared. Studies with antibody against the binding protein for Sarcophaga lectin showed that these two binding proteins are different. Since these two lectins are both active in the lectin-dependent macrophage-mediated cytotoxic reaction with Ehrlich ascites tumor cells as target cells, there must be multiple proteins on the surface of target cells that can trigger cytolytic reaction in response to different lectins in the presence of macrophages.

Participation of tumor killing factor in the antitumor effect of Sarcophaga lectin.

Antibody against tumor-killing factor inhibited cytotoxic activities produced in vitro by mouse peritoneal macrophages and in vivo in the serum of tumor-bearing mice in response to Sarcophaga lectin. These results suggest that tumor-killing factor participates in the antitumor effect of Sarcophaga lectin.

Multiple functions of general transcription factors TFIIE and TFIIH in transcription: possible points of regulation by trans-acting factors.

General transcription factors together with RNA polymerase II assemble on the promoter DNA and initiate transcription accurately in response to a variety of signals. Such signals enhance preinitiation complex formation by targeting components thereof via several alternative pathways. Two components of the initiation complexes, TFIIE and TFIIH, are known to function at both a late stage of transcription initiation and the following promoter clearance. TFIIH has been studied extensively because of its multiple enzymatic activities, functioning not only in transcription but also in nucleotide excision repair and cell cycle control. Fewer data have been reported for TFIIE, but its potential regulatory function as to TFIIH warrants further attention. In this review, an overall perspective of the functional roles of TFIIE and TFIIH during transcription initiation and the following promoter clearance will be presented as it has emerged from recent studies.

Identification and characterization of Sarcophaga lectin receptor on the surface of murine macrophages by use of monoclonal antibodies.

The structure of Sarcophaga lectin receptor on the surface of murine macrophages was analyzed using monoclonal antibodies. This receptor was found by gel filtration to have a molecular weight of 460 kDa. SDS-polyacrylamide gel electrophoresis showed that this receptor consists of two subunits of 170 kDa and 110 kDa. The results indicated that it is probably a heterotetramer of two molecules of each subunit. Two monoclonal antibodies recognized epitopes in the 110 kDa subunit, and one of them specifically inhibited the binding of Sarcophaga lectin to macrophages and the cytotoxic reaction mediated by this lectin in the presence of macrophages. Therefore, it is likely that the 110 kDa protein in the receptor plays a role in activation of macrophages by this lectin.

Transcriptional activation domain of human BTEB2, a GC box-binding factor.

BTEB2 is a GC box-binding transcription factor containing proline-rich and zinc finger domains as transactivation and DNA binding domains, respectively. We have identified a small region in the proline-rich domain indispensable for its transcription-enhancing activity by transfection experiments using various expression plasmids with point mutations or small deletions in the domain. This region comprising about 10 amino acids was relatively hydrophobic and rich in proline and alanine residues. BTEB2 purified from a baculovirus expression system could enhance transcription, depending on the presence of GC boxes in the promoter region of templates in an in vitro transcription assay. BTEB2 deleted of the hydrophobic region, however, lost the transcription-enhancing activity, in confirmation of the above results. Basic transcription factors which possibly interact with BTEB2 were examined. Initiation factors, TFIIB, TFIIE beta, and TFIIF beta as well as the TATA box-binding protein (TBP) were found to interact with BTEB2 when analyzed by in vitro binding experiments, although these interactions could not be attributed to the proline-rich domain. We discussed factors which interact with and transmit the transcriptional activity of the BTEB2 activation domain to basic transcriptional machinery.

Sequence-specific DNA damage induced by carcinogenic danthron and anthraquinone in the presence of Cu(II), cytochrome P450 reductase and NADPH.

The mechanism of metal-mediated DNA damage by carcinogenic danthron (1,8-dihydroxyanthraquinone) and anthraquinone was investigated by the DNA sequencing technique using 32P-labeled human DNA fragments obtained from the human c-Ha-ras-1 protooncogene and the p53 tumor suppressor gene. Danthron caused DNA damage particularly at guanines in the 5'-GG-3', 5'-GGGG-3', 5'-GGGGG-3' sequences (damaged bases are underlined) in the presence of Cu(II), cytochrome P450 reductase and the NADPH-generating system. The DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H2O2 and Cu(I). The formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine increased with increasing concentration of danthron. On the other hand, carcinogenic anthraquinone induced less oxidative DNA damage than danthron. Electron spin resonance study showed that the semiquinone radical could be produced by P450 reductase plus NADPH-mediated reduction of danthron, while little signal was observed with anthraquinone. These results suggest that danthron is much more likely to be reduced by P450 reductase and generate reactive oxygen species through the redox cycle, leading to more extensive Cu(II)-mediated DNA damage than anthraquinone. In the case of anthraquinone, its hydroxylated metabolites with similar reactivity to danthron may participate in DNA damage in vivo. We conclude that oxidative DNA damage by danthron and anthraquinone seems to be relevant for the expression of their carcinogenicity.

Reconstitution of damage DNA excision reaction from SV40 minichromosomes with purified nucleotide excision repair proteins.

We previously constructed the cell-free nucleotide excision repair (NER) assay system with UV-irradiated SV40 minichromosomes to analyze the mechanism of NER reaction on chromatin DNA. Here we investigate the factor that acts especially on nucleosomal DNA during the damage excision reaction, and reconstitute the damage excision reaction on SV40 minichromosomes. NER-proficient HeLa whole cell extracts were fractionated, and the amounts of known NER factors involved in the column fractions were determined by immunoblot analyses. The column fractions were quantitatively and systematically replaced by highly purified NER factors. Finally, damage DNA excision reaction on SV40 minichromosomes was reconstituted with six highly purified NER factors, XPA, XPC-HR23B, XPF-ERCC1, XPG, RPA and TFIIH, as those essential for the reaction with naked DNA. Further analysis showed that the damages on chromosomal DNA were excised as the same efficiency as those on naked DNA for short incubation. At longer incubation time, however, the damage excision efficiency on nucleosomal DNA was decreased whereas naked DNA was still vigorously repaired. These observations suggest that although the six purified NER factors have a potential to eliminate the damage DNA from SV40 minichromosomes, the chromatin structure may still have some repressive effects on NER.

Matlystatins, new inhibitors of type IV collagenases from Actinomadura atramentaria. III. Structure elucidation of matlystatins A to F.

The structures of matlystatins, novel type IV collagenase inhibitors isolated from Actinomadura atramentaria, have been determined by a systematic application of homo- and heteronuclear 2D NMR and FAB-MS/MS techniques. Their structures were characterized by the presence of piperazic acid and hydroxamic acid moieties, structural motifs often seen in protease inhibitors.

Anticellular activities of human fibroblast interferon beta and human recombinant interferon gamma. Cytologic changes in a cervical cancer cell line.

The cellular changes in a human cervical squamous-cell-cancer cell line (SKG-IIIb) were investigated after treatment with human recombinant interferon gamma and human fibroblast interferon beta. Cytoplasmic changes characterized by elongation, vacuolization and blurring of cytoplasmic borders appeared relatively early in both treatment groups. After two days of treatment with interferon gamma, the cells began to aggregate and to form pearl-like structures in the center of these aggregates; frequently seen were isolated cells with an eosinophilic cytoplasm and a pyknotic nucleus, similar to keratinizing cells. After six days of treatment with interferon beta, on the other hand, more than 20% of the cells began to show nuclear changes, including remarkable pleomorphism, multilobulation and multinucleation. The differences in biologic activities between the two interferons are discussed on the basis of these observations.

[Parameter in fulminant hepatitis].

Serum complement hemolytic activities (CH 50) and plasma prothrombin time (PTT) of patients with fulminant hepatitis (FH, 11 cases of acute type and 11 cases of subacute type), severe acute hepatitis (AH-S, 9 cases) and typical acute hepatitis (AH-T, 20 cases) were examined. AH-S was diagnosed as AH patients with PTT under 40% and without hepatic coma. Survival rates were 100% in AH-S, 45.5% (5/11) in acute type of FH and 9.1% (1/11) in subacute type of FH. The mean levels of CH 50 on their admission were 42.6 U/ml in AH-T, 28.8 U/ml in AH-S and 13.1 U/ml in FH. CH50 levels in acute hepatitis decreased in parallel to the severity of the disease and correlated with PTT (r = 0.809). In many cases of FH and almost all cases of AH-S, CH50 levels changed more slowly than those of PTT. The patients with AH-S and subacute type of FH showed a prolonged PTT and the normal levels of CH 50 in their acute phase. These results indicate that CH50 and PTT could be useful markers differentiating subacute type from acute type of fulminant hepatitis.

[A 77-year-old man with gait and gaze disturbance].

We report a 77-year-old Japanese man with progressive gait disturbance. He was well until his 71 years of the age (1992), when he noted an onset of disturbance in his speech, which was followed by difficulty in using his left hand. He did not attempt to use his left hand afterwards. He started to fall down in the spring of 1994. He was admitted to our service on October 6, 1994. Neurologic examination revealed an alert and oriented man. He showed limb-kinetic apraxia in his left hand with anosognosia for his apraxia. Vertical gaze was impaired. He walked in small steps. He had moderate axial and limb rigidity. He had no weakness, ataxia, or tremor. Deep tendon reflexes were normal. Plantar response was flexor. Sensation was intact. His gait had progressively become worse and he was admitted to another hospital in April of 1996. At that time he was disoriented to time. He was only able to walk a few steps with support. He continued to show limb-kinetic apraxia in his left hand. He developed dementia and dysphagia and he expired on October 27, 1998. He was discussed in a neurological CPC, and the chief discussant arrived at the conclusion that the patient had corticobasal degeneration. Most of the participants agreed with this diagnosis, but a few of them thought that progressive supranuclear palsy would be more likely. Post-mortem examination revealed no gross cortical atrophy. The right hemisphere was kept frozen for future biochemical analysis. The left precentral gyrus showed spongy changes, neuronal loss and gliosis. The pallidum, putamen, and the subthalamic nucleus were unremarkable, however, neurofibrillary tangles were seen in the subthalamic nucleus. The substantia nigra showed only slight neuronal loss; neuronal pigments were well retained. A few neurofibrillary tangles were seen in the remaining neurons. The cerebellar dentate nucleus showed grumose degeneration. Gallyas-Braak staining revealed many tuft-shaped astrocytes in the precentral gyrus. Pathologic diagnosis was progressive supranuclear palsy. Some participants thought that this diagnosis was unacceptable, because the pathologic changes in the substantia nigra, globus pallidus, and the subthalamic nucleus, which were usually severely involved in PSP, did not show typical changes of PSP. In addition, the predominant clinical feature was limb-kinetic apraxia, although he showed vertical gaze paresis and parkinsonian gait, which could also be seen in corticobasal degeneration. There was a big discussion among participants with regard to the diagnosis.

[A 70-year-old man with right hemiparesis and mutism].

We report a 70-year-old man who had a sudden onset of right hemiparesis and mutism. The lower extremity was more involved than the upper one. He had a long history of diabetes and chronic renal failure for which hemodialysis was necessary. On August 30, 1990, he had an sudden onset of right hemiparesis and mutism. Neurological examination revealed awake but mute in no acute distress. He could only respond to very simple commands such as opening his mouth or protruding his tongue. He did not appear to understand more difficult questions. In addition, he could not answer verbally. He was totally mute. Cranial nerves appeared intact except for slight right central facial paresis and severe diabetic retinopathy. He had complete paralysis of his right leg and a moderate weakness in his right upper extremity. Deep reflexes were diminished in both upper extremities and absent in the lower limbs. Frotal signs such as grasp and snout reflexes were present. Cranial CT scans revealed an ill-defined low density area in the left parasagittal subcortical area and a part of the anterior cerebral artery territory. The supplementary motor area appeared at least in part to be involved. He was treated with glycerol and other supportive cares, however, his clinical course was complicated by pneumonia, heart failure, septicemia, and he expired two months after his stroke. The patient was discussed in a neurological CPC, and the chief discussant arrived at a conclusion that he had an artery-to-artery embolism at the internal carotid bifurcation resulting in the cerebral infarction mainly in the territory of the anterior cerebral artery.(ABSTRACT TRUNCATED AT 250 WORDS)

[A 29-year-old man with diabetes insipidus and cerebellar ataxia and development of spinal cord swelling 15 years after the onset].

We report a 29-year-old man with diabetes insipidus and cerebellar ataxia who developed spinal cord swelling 15 years after the onset. He was well until 14 years of the age when he noted dizziness. Two years after there was an onset of gait disturbance and slurred speech. He also noted polydipsia and polyuria. He was evaluated at the neurosurgery service of our hospital when he was 17 years of the age. Neurologic examination at that time revealed memory loss, horizontal nystagmus, cerebellar ataxic gait, dysmetria and decomposition more on the left. Cranial CT scan revealed a mass lesion involving the left subthalamic region and the head of the caudate area. Spinal fluid was unremarkable, however, human chorionic gonadotropin was increased to 27 mIU/ml. He was treated by radiation therapy (3,000 rads for total brain area and 5,460 rads for focal region). His CT scan and memory loss improved, however, cerebellar ataxia was unchanged. Three years after the radiation, he started to show choreic movement in his neck and left upper extremity. He was admitted to our service in August 14, 1995 when he was 29 years of the age. On admission, he was alert but disoriented to time; calculation was also poor. Higher cerebral functions were intact. The optic fundi were normal without papilledema. Visual field appeared intact. Gaze nystagmus was observed in all the directions, but more prominent in the horizontal direction. Speech was slurred. Otherwise, cranial nerves were unremarkable. Motor wise, he showed marked truncal and gait ataxia; he was unable to walk because of ataxia. Muscle atrophy and marked weakness was noted in both upper extremities more on the left side. Deep tendon reflexes were diminished in the upper extremities but active in the lower extremities. He was polyuric; urinary specific gravity was low. Spinal fluid contained 6 cells/cmm and 113 mg/ dl of protein; Queckenstedt was positive. MRI revealed swelling of the cervical cord; in addition, the entire cervical region and the medullar oblongata appeared as high signal intensity areas. No mass lesion was noted in the supratentorial structures but the third ventricle was markedly enlarged. Surgical biopsy was performed on the cervical lesion. The patient was discussed in neurologic CPC, and the chief discussant arrived at the conclusion that the patient had germinoma with syncytiotrophoblastic giant cells in the diencephalic region which appeared to have been cured by radiation therapy; he thought that the cervical lesion was the seeding of germinoma. Cerebellar ataxia was ascribed to the remote effect of germinoma. Most of the participants thought that the original tumor was germinoma and the cervical lesion was its spread. Some participants thought that his ataxia was caused by germinoma cells involving the medulla and the inferior cerebellar peduncles. Histologic observation of the biopsied tissue from the spinal cord revealed the typical two cell patterned germinoma. Most of the tumor cells were not stained for an antibody against HCG, but some tumor cells were positively stained. Germinoma is very radio-sensitive; this patient showed T2 high signal lesion involving the medulla oblongata and cervical cord continuously. Probably, tumor cells in the lower brain stem escaped radiation, and gradually spread to the spinal cord over many years. At the time of operation, the surface of the spinal cord was free from tumor cells. Therefore, tumor cells invaded the spinal cord continuously from the medulla oblongata. He was treated with cervical radiation, and his neurologic as well as radiologic findings showed marked improvement.

[A 40-year-old woman with progressive dementia and abnormal behavior].

We report a 40-year-old Japanese woman who died after 12 years history of progressive dementia and abnormal behaviors. She was well until 1985 at her age of 28 years old, when she had an onset of behavioral change in which she drank much, neglected house-keeping works, and her life style became sloppy. At age 30, she became unable to understand written sentences, and paced up- and down in and out of her house. She was admitted to other hospital where marked dementia with disorientation and memory loss were noted. Slight increase in CSF protein and decrease in the peripheral nerve conduction velocity were also noted at that time. In the next year, she started to have convulsions. These symptoms had progressively become worse and was admitted to Tokyo Metropolital Matsuzawa Hospital in June of 1991 when she was 34 years of age. Despite marked dementia, she was able to walk normally, no motor paralysis, cerebellar ataxia, nor dyskinesia were noted. Deep tendon reflexes were diminished. MRI revealed T-2 high signal intensity lesions involving the white matter of the cerebrum predominantly in the frontal region. In about one year, she started to show difficulty in gait, and she became bed-ridden in July of 1994. She was discharged to home for a while, but required admission again. She expired on February 5, 1998. Her younger brother had an essentially similar dementing disease and he expired at the age of 35 years. The parents were of first cousins. The patient was discussed in a neurological CPC, and the chief discussant arrived at the conclusion that the patient had adult form of metachromatic leukodystrophy, because of white matter change in the frontal lobe, decrease in nerve conduction velocity, convulsion, marked dementia, and consanguineous marriage with a similarly affected brother. Most of the audience agreed with this conclusion, but the differential diagnosis from globoid cell leukodystrophy was felt difficult from the clinical findings alone. Post-mortem examination revealed marked atrophy in the frontal lobe. Cerebellum appeared to be smaller than normal. In the coronal sections, marked atrophy of the white matter with brown discoloration was noted. The lateral ventricles were dilated. Klüver-Barrera staining revealed marked demyelination with relative preservation of the U-fibers. PAS-positive materials were deposited in some astrocytes as well as neurons. Metachromatic deposits were noted not only in the cerebrum but also cerebllum after staining with acid cresyl violet. Pathologic diagnosis was consistent with adult type of metachromatic leukodystrophy.

[MAC-awake and wake-up time of isoflurane and sevoflurane with reference to the concentration of gas, duration of inhalation and patient's age and obesity].

We evaluated the influence of the concentration of volatile anesthetics, the duration of inhalation time, the patient's age and degree of obesity on MAC-awake (the end-tidal concentration of volatile anesthetics on awakening) and Wake-up time (the period from stopping inhalation to eye-opening in response to verbal command) following isoflurane (Iso) or sevoflurane (Sev) anesthesia in 240 patients (ASA I or II, age 17-84 yr). The patients were anesthetized with 50% oxygen, 50% nitrous oxide and various concentrations of Iso or Sev. They were divided into 6 groups in respect to the end-tidal concentration of Iso or Sev: Iso 0.7% (1 MAC), Iso 1.0% (1.5 MAC), Iso 1.4% (2 MAC), Sev 0.9% (1 MAC), Sev 1.3% (1.5 MAC) and Sev 1.8% (2 MAC). After operation all anesthetics were discontinued and MAC-awake and Wake-up time were measured under 100% oxygen inhalation. MAC-awake value of Iso was 0.14 +/- 0.05% (SD)% in all groups and that of Sev was 0.17 +/- 0.05% in Sev 0.9% group, 0.16 +/- 0.05% in Sev 1.3%, 0.17 +/- 0.06% in Sev 1.8%, respectively. All of them became smaller in aged groups than in younger groups but they were not influenced by the concentration of gas, the duration of inhalation nor the degree of obesity.(ABSTRACT TRUNCATED AT 250 WORDS)