[International expert consensus on gene therapy for hereditary hearing loss: based on clinical trials].
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Journal Article↗
Biomedical subjects
Y Tao
Publications and source records attributed to Y Tao.
The human homologue of Drosophila TRF-proximal protein is associated with an RNA polymerase II-SRB complex.
Mammalian RNA polymerase II holoenzymes are large complexes that have been reported to contain, in addition to RNA polymerase II, homologues of several yeast SRBs, various general transcription factors, and other polypeptides. On the basis of its copurification with an SRB-containing RNA polymerase II complex by conventional chromatography procedures, we have identified a human homologue of Drosophila TRF-proximal protein, designated hTRFP, and isolated its cognate cDNA. Antibody specific for SRB7 can immunoprecipitate hTRFP and RNA polymerase II and, reciprocally, antibody specific for hTRFP can immunoprecipitate RNA polymerase II and SRB7. These data indicate that hTRFP is an integral component of an RNA polymerase II-SRB complex. Whereas the precise function of hTRFP remains to be determined, the hTRFP-containing RNA polymerase II-SRB complex supports basal level transcription and, relative to RNA polymerase II alone, enhances transcriptional activation by Gal4-VP16 in the presence of cofactor PC4. Thus, hTRFP may regulate transcription of class II genes through association with the RNA polymerase II-SRB complex.
Amino Acid Sequence↗
Assembly of a tailed bacterial virus and its genome release studied in three dimensions.
We present the first three-dimensional reconstruction of a prolate, tailed phage, and its empty prohead precursor by cryo-electron microscopy. The head-tail connector, the central component of the DNA packaging machine, is visualized for the first time in situ within the Bacillus subtilis dsDNA phage phi29. The connector, with 12- or 13-fold symmetry, appears to fit loosely into a pentameric vertex of the head, a symmetry mismatch that may be required to rotate the connector to package DNA. The prolate head of phi29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T=3 quasi-symmetry. Reconstruction of an emptied phage particle shows that the connector and neck/tail assembly undergo significant conformational changes upon ejection of DNA.
Bacillus Phages↗
Structure of bacteriophage T4 fibritin M: a troublesome packing arrangement.
Fibritin, a 52 kDa product of bacteriophage T4 gene wac, forms 530 A long fibers, named whiskers, that attach to the phage neck and perform a helper function during phage assembly. Fibritin is a homotrimer, with its predominant central domain consisting of 12 consecutive alpha-helical coiled-coil segments linked together by loops. The central domain is flanked by small globular domains at both ends. Fibritin M is a genetically engineered fragment of the wild type and contains 74 amino-acid residues corresponding to the last coiled-coil segment and the complete carboxy-terminal domain. The crystals of fibritin M belong to the rare space group P3 with three crystallographically independent trimers in the unit cell. The structure has been established at 1.85 A resolution by combining molecular and isomorphous replacement techniques. One of the two heavy-atom derivatives used was gaseous xenon. A substantial fraction of residues in each independent trimer is disordered to various extents in proportion to the lack of restraints on the molecules provided by the lattice contacts. Accurate modeling of the solvent present in the crystals was crucial for achieving good agreement with experimental data.
Amino Acid Sequence↗
Analysis of a distal cluster of binding elements and other unusual features of the promoter of the highly regulated nit-3 gene of Neurospora crassa.
In Neurospora crassa, the expression of the nit-3 gene (nitrate reductase) is dependent upon nitrogen derepression and nitrate induction and is regulated by two positive-acting transcription factors, NIT2 and NIT4, and a negative regulator, NMR. The presence of a tightly linked cluster of NIT2 and NIT4 binding sites suggested that their close spacing might be required for a synergistic interaction of the NIT2 and NIT4 proteins. We show here that the NIT2 and NIT4 binding sites can be separated without affecting either the expression level or the precise regulation of the nit-3 gene. Studies conducted on the NIT2 site II, which contains only a single GATA element and yet plays a major role in nit-3 gene expression, showed that nucleotides both 5' and 3' of the GATA sequence were important for strong DNA binding in vitro and its activation function in vivo. The nit-3 promoter contains two long AT-rich sequences, one of which is located just upstream of the transcription start sites and is required for optimal promoter function. The nit-3 transcript contains eight TACC repeats in its 5' noncoding region which appear to be involved in mRNA instability. Deletion of these TACC repeats led to a significant increase in the stability of nit-3 mRNA.
Binding Sites↗
Genomics and transcription analysis of human TFIID.
TFIID, a multisubunit protein comprised of TBP (TATA box-binding protein) and TAF(II)s (TBP-associated factors), has a central role in transcription initiation at class II promoters. TAF(II)s role as mediators of regulatory transcription factors, such as pRb and p53, and their involvement in signal transduction pathways suggest that some may participate in the control of cell proliferation and differentiation: therefore, they could be considered potential protooncogenes or antioncogenes. With the aim of starting to analyse these potential roles, we have determined the genomic position of nine human TAF(II) genes (TAF[II]250, TAF[II]135, TAF[II]100, TAF[II]80, TAF[II]55, TAF[II]43, TAF[II]31, TAF[II]28, TAF[II]20/15) and of two previously unknown sequences related to TAF(II)250 and TAF(II)31, respectively. Except for those encoding TAF(II)250 and TAF(II)31, these genes are present in a single copy and, with the exclusion of those for TAF(II)43 and TAF(II)28 (both at 6p21), are localized in different segments of the genome. Indeed, six of them map to a chromosomal region commonly altered in specific neoplasias, which defines them as candidates for involvement in oncogenesis. Our experiments also demonstrate that TAF(II) transcripts are synthesized ubiquitously, mostly at low levels similar to those of TBP. Interestingly, the amount of the major mRNA species detected by TAF(II)20/15 cDNA is higher, which suggests that the polypeptide it encodes may also perform functions independently of TFIID. TAF(II) isoforms, indicated by additional bands on Northern blots, may play a role in modulation of TFIID function. These data will be useful for analysing variations of TAF(II) mRNA phenotype during cell proliferation, differentiation and development, both normal and pathological.
Animals↗
A cofactor, TIP30, specifically enhances HIV-1 Tat-activated transcription.
Replication of HIV-1 requires the viral Tat protein, which increases the extent of transcription elongation by RNA polymerase II after activation at the single viral long terminal repeat (LTR) promoter. This effect of Tat on transcription requires Tat interactions with a 5' region (TAR) in nascent transcripts as well as Tat-specific cofactors. The present study identifies a cellular protein, TIP30, that interacts with Tat and with an SRB-containing RNA polymerase II complex both in vivo and in vitro. Coexpression of TIP30 specifically enhances transactivation by Tat in transfected cells, and immunodepletion of TIP30 from nuclear extracts abolishes Tat-activated transcription without affecting Tat-independent transcription. These results implicate TIP30 as a specific coactivator that may enhance formation of a Tat-RNA polymerase II holoenzyme complex.
Acetyltransferases↗
Enhanced gamma-glutamyl transpeptidase expression and selective loss of CuZn superoxide dismutase in hepatic iron overload.
Liver injury caused by iron overload is presumed to involve lipid peroxidation and the formation of products such as 4-hydroxynonenal (4HNE), which has been implicated in hepatic fibrogenesis. Cellular antioxidants that modulate the formation and detoxification of compounds such as 4HNE may represent important protective mechanisms involved in the response to iron overload. This study examines the relationship between 4HNE, collagen content, and antioxidant defenses in the livers of rats fed carbonyl iron for 10 weeks. Iron-loading resulted in significant increases in iron (8.8-fold), 4HNE (1.7-fold), and hydroxyproline (1.5-fold). Total glutathione content was unchanged by iron, but gamma-glutamyl transpeptidase activity (GGT) increased sixfold and CuZn superoxide dismutase (CuZnSOD) activity decreased >9%. GGT colocalized with iron deposition and was associated with increased GGT mRNA. Decreased CuZnSOD activity was paralleled by a reduction in CuZnSOD protein on Western blot and immunohistochemistry, but no decrease in CuZnSOD mRNA. Glutathione S-transferase (GST) and Mn superoxide dismutase (MnSOD) activities were also significantly increased by iron loading. These results demonstrate that iron overload significantly alters the expression of antioxidant enzymes associated with glutathione (GGT and GST) and superoxide metabolism (CuZnSOD and MnSOD). Furthermore, the localized induction of GGT may enhance detoxification of lipid peroxidation-derived aldehydes via glutathione-dependent pathways in iron-loaded hepatocytes. These alterations in antioxidant defenses may represent an adaptive response, limiting accumulation 4HNE, and thus, stimulation of collagen synthesis, accounting for the mild fibrogenic response seen in this model of iron overload.
Aldehydes↗
Transcription activation via enhanced preinitiation complex assembly in a human cell-free system lacking TAFIIs.
In contrast to previous findings in cell-free systems reconstituted with partially purified metazoan factors, we demonstrate dramatic activation of transcription in a TBP-dependent but TAFII-independent manner in HeLa nuclear extracts immunodepleted of TBP and major TAFIIs. Single-round transcription assays reveal that TAFII-independent activation is manifested at the level of productive preinitiation complex formation and that TAFIIs actually impair functional preinitiation complex assembly in a core promoter-specific manner. Furthermore, TAFIIs appear to elevate absolute levels of transcription under multiple-round transcription conditions, presumably by facilitating secondary initiation events. Finally, human coactivator activities related to those in yeast RNA polymerase II/mediator complexes appear to function in unfractionated HeLa nuclear extracts.
Cell Extracts↗
Synthesis and differential turnover of the CYS3 regulatory protein of Neurospora crassa are subject to sulfur control.
The transcription factor CYS3 of Neurospora crassa is a positive regulator of the sulfur regulatory circuit which contains many structural genes involved in sulfur metabolism. Expression and degradation of the CYS3 protein are precisely regulated in a sulfur-dependent manner. cys-3 expression was found to be fully repressed by high concentrations of methionine or inorganic sulfate present in the culture medium and to be derepressed when these favored sulfur sources were limited. cys-3 transcripts could be readily detected within 2 h after derepression, whereas the CYS3 protein was not found until after 4 h. CYS3 is stable, with a half-life greater than 4 h under low-sulfur conditions when it is required for cell growth. However, it is degraded relatively quickly when methionine or inorganic sulfate becomes available. Upon sulfur repression, cys-3 transcripts disappeared within 30 min with an estimated half-life of 5 min whereas CYS3 protein almost entirely disappeared in 1 h with a half-life of approximately 10 min. These results suggest that a selective elimination of CYS3 is a highly regulated process. Site-directed mutagenesis showed that Lys-105 of CYS3 is important for its instability. The change of this single residue from lysine to glutamine resulted in a prolonged half life of CYS3 and impaired responsiveness of CYS3 degradation to sulfur level changes.
Cystathionine gamma-Lyase↗
Novel cofactors and TFIIA mediate functional core promoter selectivity by the human TAFII150-containing TFIID complex.
TATA-binding protein-associated factors (TAFIIs) within TFIID control differential gene transcription through interactions with both activators and core promoter elements. In particular, TAFII150 contributes to initiator-dependent transcription through an unknown mechanism. Here, we address whether TAFIIs within TFIID are sufficient, in conjunction with highly purified general transcription factors (GTFs), for differential core promoter-dependent transcription by RNA polymerase II and whether additional cofactors are required. We identify the human homologue of Drosophila TAFII150 through cognate cDNA cloning and show that it is a tightly associated component of human TFIID. More importantly, we demonstrate that the human TAFII150-containing TFIID complex is not sufficient, in the context of all purified GTFs and RNA polymerase II, to mediate transcription synergism between TATA and initiator elements and initiator-directed transcription from a TAFII-dependent TATA-less promoter. Therefore, TAFII-promoter interactions are not sufficient for the productive core promoter-selective functions of TFIID. Consistent with this finding, we have partially purified novel cofactor activities (TICs) that potentiate the TAFII-mediated synergism between TATA and initiator elements (TIC-1) and TAFII-dependent transcription from TATA-less promoters (TIC-2 and -3). Furthermore, we demonstrate an essential function for TFIIA in TIC- and TAFII-dependent basal transcription from a TATA-less promoter. Our results reveal a parallel between the basal transcription activity of TAFIIs through core promoter elements and TAFII-dependent activator function.
ARNTL Transcription Factors↗
Relationship between inflammatory cells in bronchoalveolar lavage fluid and pathologic changes in the lung interstitium.
Bronchoalveolar lavage (BAL) is recognized as an important research tool for various lung diseases, but it is still uncertain whether inflammatory cells in BAL fluid (BALF) accurately reflect pathologic changes in the lung interstitium. We used a morphometric method to quantify the density of inflammatory cells in the lung interstitium by utilizing a computer-aided graphic analyzer and compared those findings with BALF results. Two types of animal models were studied, i.e., endotoxemia (Escherichia coli endotoxin) and hypersensitivity pneumonitis (inhaled ovalbumin). Male Wistar rats were used; the right lungs were lavaged and the left lungs were prepared for morphometric study. In the endotoxemia model, the neutrophil fraction in BALF and the neutrophil density in the lung interstitium correlated significantly at 18 h (r = 0.81, p < 0.05) and 24 h (r = 0.81, p < 0.05) but not at any other time points after injection. In the hypersensitivity pneumonitis model, the neutrophil fraction in BALF and the neutrophil density in the lung interstitium correlated significantly (r = 0.80, p < 0.05) only at 3 h after inhalation. The lymphocyte fraction in BALF and the lymphocyte density in the lung interstitium were correlated positively at 3 h (r = 0.83, p < 0.05), 1 day (r = 0.82, p < 0.05), 2 days (r = 0.67, p = NS), and 4 days (r = 0.87, p < 0.05), but not at 6 days after inhalation. Our data suggest that neutrophil fraction in BALF does not reflect neutrophil populations in the lung interstitium except at the time of maximal neutrophil count in lung lavage. For lymphocytes in the hypersensitivity pneumonitis model, those in BALF and in the lung interstitium roughly correlate in the majority of measurements.
Alveolitis, Extrinsic Allergic↗
Modulation of stress protein (hsp27 and hsp70) expression in CD4+ lymphocytic cells following acute infection with human immunodeficiency virus type-1.
This study was designed to assess the impact of acute human immunodeficiency virus (HIV-1) infection on host intracellular expression of the heat shock family of stress proteins (hsps). Experimental conditions were established wherein CD4+ lymphocytic cell lines undergo a synchronous HIV-1 infection cycle. During the early phase of infection, HIV-1 mRNA expression was restricted to singly and multiply spliced subspecies, with no genomic viral RNA present until 30 hr following infection. In contrast, hsp27 and hsp70 mRNA transcription appeared as early as 3-8 hr following viral infection. No corresponding induction was observed in mock-infected cells. Notably, hsp27 and hsp70 mRNA transcripts were down-regulated by 24 hr, concomitant to the first appearance of full-length genomic HIV-1 mRNA. Hsp27 and hsp70 mRNA transcripts reemerged at end stages of the viral replicative cycle, coincident to virion release and CD4 cell death. Similarly, a transient induction of de novo hsp27 protein expression occurred between 12 and 24 hr. The generated hsp27 stress response was viral dose-related, suppressed by heat-inactivation of virus, and abrogated by neutralizing antibodies to HIV-1. Acute infection did not alter levels of hsp60, hsp70, and hsp90 protein synthesis. However, two-dimensional Western blot analysis did show the appearance of novel hsp70 homologues between 6 and 24 hr following infection. CEM.NKR, Jurkat, H9, and MT-2 cells showed similar patterns of viral-associated modulation of host hsp27 and hsp70 protein and RNA expression. Thus, host hsp27 and hsp70 stress pathways are selectively implicated in the HIV-1 viral life cycle.
Blotting, Northern↗
Structure of bacteriophage T4 fibritin: a segmented coiled coil and the role of the C-terminal domain.
BACKGROUND: Oligomeric coiled-coil motifs are found in numerous protein structures; among them is fibritin, a structural protein of bacteriophage T4, which belongs to a class of chaperones that catalyze a specific phage-assembly process. Fibritin promotes the assembly of the long tail fibers and their subsequent attachment to the tail baseplate; it is also a sensing device that controls the retraction of the long tail fibers in adverse environments and, thus, prevents infection. The structure of fibritin had been predicted from sequence and biochemical analyses to be mainly a triple-helical coiled coil. The determination of its structure at atomic resolution was expected to give insights into the assembly process and biological function of fibritin, and the properties of modified coiled-coil structures in general. RESULTS: The three-dimensional structure of fibritin E, a deletion mutant of wild-type fibritin, was determined to 2.2 A resolution by X-ray crystallography. Three identical subunits of 119 amino acid residues form a trimeric parallel coiled-coil domain and a small globular C-terminal domain about a crystallographic threefold axis. The coiled-coil domain is divided into three segments that are separated by insertion loops. The C-terminal domain, which consists of 30 residues from each subunit, contains a beta-propeller-like structure with a hydrophobic interior. CONCLUSIONS: The residues within the C-terminal domain make extensive hydrophobic and some polar intersubunit interactions. This is consistent with the C-terminal domain being important for the correct assembly of fibritin, as shown earlier by mutational studies. Tight interactions between the C-terminal residues of adjacent subunits counteract the latent instability that is suggested by the structural properties of the coiled-coil segments. Trimerization is likely to begin with the formation of the C-terminal domain which subsequently initiates the assembly of the coiled coil. The interplay between the stabilizing effect of the C-terminal domain and the labile coiled-coil domain may be essential for the fibritin function and for the correct functioning of many other alpha-fibrous proteins.
Amino Acid Sequence↗
Specific interactions and potential functions of human TAFII100.
Human transcription initiation factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFs). To investigate the structural organization and function of TFIID, we have cloned and expressed a DNA encoding the third largest human TFIID subunit, hTAFII100. Immunoprecipitation studies demonstrate that hTAFII100 is an integral subunit that is associated with all transcriptionally-competent forms of TFIID. They further suggest that at least part of the N-terminal region lies on the surface of TFIID, while a C-terminal region containing conserved WD-40 repeats appears inaccessible. Both in vivo and in vitro assays indicate that hTAFII100 interacts strongly with the histone H4-related hTAFII80 and the histone H3-related hTAFII31, as well as a stable complex comprised of both hTAFII80 and hTAFII31. Apparently weaker interactions of hTAFII100 with TBP, hTAFII250, hTAFII28, and hTAFII20, but not hTAFII55, also have been observed. These results suggest a role for hTAFII100 in stabilizing interactions of TAFs, especially the histone-like TAFs, in TFIID. In addition, functional studies show that anti-hTAFII100 antibodies selectively inhibit basal transcription from a TATA-less initiator-containing promoter, relative to a TATA-containing promoter, suggesting a possible core promoter-specific function for hTAFII100.
Amino Acid Sequence↗
Randomized trial of breast self-examination in Shanghai: methodology and preliminary results.
BACKGROUND: The efficacy of breast self-examination in helping to reduce mortality from breast cancer has not been rigorously demonstrated. PURPOSE: To assess efficacy, a large, randomized trial was initiated in Shanghai, China. METHODS: From October 1989 to October 1991, 267040 current and retired female employees associated with 520 factories in the Shanghai Textile Industry Bureau were randomly assigned on the basis of factory to either a self-examination instruction group (133375 women) or a control group (133665 women). The women were born within the period from 1925 through 1958. Women in the instruction group were given intensive training in breast self-examination, including the use of silicone breast models and personalized instruction, plus two subsequent reinforcement sessions and multiple reminders to practice the technique. Women in the control group were asked to attend training sessions on the prevention of low back pain. All women have been followed for the development of breast diseases and for death from breast cancer. RESULTS: A high level of participation during the first 4-5 years of the trial was documented among women in the instruction group. Randomly sampled women in this group demonstrated greater proficiency in detecting lumps in breast models than did randomly sampled women in the control group. Approximately equal numbers of breast cancers were detected in the two groups (331 in the instruction group and 322 in the control group) through 1994, which is the last year for which case-finding efforts have been completed. The breast cancers detected in the instruction group were not diagnosed at an appreciably earlier stage or smaller size than those in the control group. More benign breast lesions were detected in the instruction group than in the control group (1457 versus 623, respectively), suggesting a higher index of suspicion for women who received training. Cumulative breast cancer mortality rates through 5 years from entry into the study were nearly equivalent for the two groups. CONCLUSIONS: Breast self-examination has not led to a reduction in mortality from breast cancer in this study cohort in the first several years since the trial began. A shift toward the diagnosis of disease at a less advanced stage in women given instruction has also not been demonstrated. Longer follow-up of participants in this trial is required before final assessment can be made of the efficacy of breast self-examination. IMPLICATIONS: At this time, there is insufficient evidence to recommend for or against the teaching of breast self-examination.
Adult↗
Acylation-stimulating protein (ASP) regulates glucose transport in the rat L6 muscle cell line.
Acylation-stimulating protein (ASP), a human plasma protein, is a potent stimulator of triglyceride synthesis and glucose transport in both human adipocytes and fibroblasts. The purpose of the present in vitro study was to examine the effect of ASP on glucose transport in muscle cells. ASP stimulated 2-deoxy-glucose transport (2-DG) in differentiated rat L6 myotubes in a time (30 min to 24 h) and concentration dependent manner (97% increase). The magnitude of the ASP effect on glucose transport was comparable to the time- and concentration-dependent effects seen with insulin (125% increase), but was additive to insulin, pointing to involvement of differential signalling pathways. ASP stimulation was dependent on cell differentiation in that glucose transport increased by only 12% in myoblasts, comparable to the effect of insulin in myoblasts (15% increase) demonstrating selective responsiveness of the differentiated myotubes to ASP and insulin. The mechanism for the ASP induced increase in glucose transport was also examined. ASP increased the Vmax for 2-DG transport by 183% (4.02 vs. 1.42 nmol/mg cell protein/30 s; ASP vs. Control, respectively). This could be explained by an increased translocation of glucose transporters (GLUT 1, GLUT 4 and GLUT 3) to the plasma membrane surface as demonstrated by Western analysis (+43% P < 0.05, +30% P < 0.05, and +49% P < 0.05, respectively). The effects of ASP were equal to those of insulin (+47%, +26% and +53% for GLUT 1, GLUT 4 and GLUT 3, respectively) and in all cases were paralleled by comparable glucose transport increases under the same incubation conditions. After long-term stimulation (24 h), Western analysis indicated that ASP had a permissive effect on insulin stimulated increases in total GLUT3 and GLUT4 cellular transporter content. These results suggest that muscle is also responsive to ASP and that ASP may play a role in glucose metabolism in both muscle and adipose tissue.
Animals↗
Pain as a predictor of outcome in patients with operable pancreatic carcinoma.
BACKGROUND: The purpose of our study was to evaluate the relationship between pain and resectability and survival in patients with operable pancreatic carcinoma. METHODS: Pain, pain intensity, and pain location were prospectively assessed in newly diagnosed patients with operable adenocarcinoma of the pancreas. Patients were evaluated before their first operation at a large tertiary care cancer center. Pain factors were then correlated with outcomes of surgery, including resectability and survival. RESULTS: Seventy-seven patients with operable pancreatic carcinoma were evaluated before operation. With the Memorial Pain Assessment Card and a demographic questionnaire, an analysis of analgesic use and pain prevalence and intensity were quantitated. Twenty-two (29%) of 77 patients reported no pain. Fifty-five had mild to severe pain. Moderately severe or greater pain (Memorial Pain Assessment Card Tursky scores of 5 or greater or visual analogue self-assessment pain intensity scores greater than 30) was found in 20 patients. Twenty-six (34%) patients had resectable disease. Of the 51 patients who did not have resections, 35 had metastatic disease. Locoregional unresectable disease without metastases was found in 16 patients. Resectability was correlated with the presence of pain (p = 0.04). The median survival for all patients was 6.7 months. Not surprisingly, patients undergoing resection had a significantly better median survival than did those whose disease was unresectable (5.5 versus 15.1 months). Pain before operation significantly predicted survival (median survival for those with pain, 5.7 months; for those without pain, 15 months; p = 0.003). Even among patients who underwent resection, the presence of pain was associated with a worse survival (21.9 months versus 9.2 months; p = 0.045). In a multivariate analysis the two significant variables were inability to undergo resection and presence of any pain. CONCLUSIONS: The presence of pain in newly diagnosed patients with potentially operable pancreatic cancer is an ominous predictor of resectability and of survival. Even if the patient can undergo resection, the presence of preoperative pain is associated with a poor prognosis. Patients with operable pancreatic cancer who present with pain, even those whose evaluation shows a likelihood of resectability, are at high risk for recurrence with an impaired survival compared with those patients without pain.
Adenocarcinoma↗