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PubMed1994

Transcriptional regulation of the apoAI gene by hepatic nuclear factor 4 in yeast.

Hepatocyte Nuclear Factor 4 (HNF-4), a liver-enriched orphan receptor of the nuclear receptor superfamily, is required for the expression of a wide variety of liver-specific genes including apoAI. To explore the possibility that site A of the apoAI gene enhancer might also be the target for HNF-4 without the interference of endogenous mammalian cell proteins that also bind to site A, we tested the ability of HNF-4 to activate transcription from site A in yeast cells. Electrophoretic mobility shift assays (EMSA) and Scatchard plot analysis demonstrated that yeast produced HNF-4 binds to site A with an affinity two times higher than that of yeast produced RXR alpha. Mapping analysis indicated that the 5' portion of site A containing two imperfect direct repeats (TGAACCCTTGACC) and the sequence of the trinucleotide spacer (CCT) between these imperfect repeats are critical determinants for selective binding and transactivation by HNF-4. Similar observations were obtained when these mutated versions of site A were evaluated by transient cotransfection assays in CV1 cells. We conclude that the unique structural determinants of site A in conjunction with the differential binding affinity of HNF-4 for site A may play a fundamental role in apoAI gene regulation.

Animals↗
PubMed2000

CArG binding factor A (CBF-A) is involved in transcriptional regulation of the rat Ha-ras promoter.

In the present study we identified a positive transcriptional element within the rat Ha-ras promoter previously known as Ha-ras response element (HRE) and identified a trans-acting factor that binds HRE sequences in rat mammary cells. To identify the binding protein we employed sequence specific DNA affinity chromatography. Amino acid sequence analysis of the affinity-purified proteins was performed by tandem mass spectroscopy. The results unexpectedly demonstrated that in rat mammary cells CArG box-binding factor A (CBF-A) is the major protein species that bind specifically to the rat and human HRE sequences with high affinity. The affinity of CBF-A binding to HRE was significantly higher than to the CArG box described as a recognition sequence for CBF-A protein. Transient transfection assays using reporter plasmids verified that mutations within the HRE that disrupt binding of CBF-A also reduced the activity of the rat Ha-ras promoter. Despite the fact that the HRE within the Ha-ras promoter resembles a binding site for Ets transcription factors, we did not detect the binding of Ets-related proteins to the rat HRE in BICR-M1Rk cells. We further demonstrated a correlation between the presence of HRE binding activity and induction of Ha-ras mRNA expression following serum stimulation in the mammary carcinoma cell line. Taken together, our results suggest that CBF-A may play an important role in transcriptional regulation of Ha-ras promoter activity during normal mammary cell growth and carcinogenesis.

Animals↗
PubMed2003

Transcriptional activation of immediate-early gene ETR101 by human T-cell leukaemia virus type I Tax.

Human T-cell leukaemia virus type I (HTLV-I) Tax regulates viral and cellular gene expression through interactions with multiple cellular transcription pathways. This study describes the finding of immediate-early gene ETR101 expression in HTLV-I-infected cells and its regulation by Tax. ETR101 was persistently expressed in HTLV-I-infected cells but not in HTLV-I uninfected cells. Expression of ETR101 was dependent upon Tax expression in the inducible Tax-expressing cell line JPX-9 and also in Jurkat cells transiently transfected with Tax-expressing vectors. Tax transactivated the ETR101 gene promoter in a transient transfection assay. A series of deletion and mutation analyses of the ETR101 gene promoter indicated that a 35 bp region immediately upstream of the TATA-box sequence, which contains a consensus cAMP response element (CRE) and a G+C-rich sequence, is the critical responsive element for Tax activation. Site-directed mutagenesis analysis of the 35 bp region suggested that both the consensus CRE motif and its upstream G+C-rich sequence were critical for Tax transactivation. Electrophoretic mobility shift analysis (EMSA) using the 35 bp sequence as probe showed the formation of a specific protein-DNA complex in HTLV-I-infected cell lines. EMSA with specific antibodies confirmed that the CREB transcription factor was responsible for formation of this specific protein-DNA complex. These results suggested that Tax directly transactivated ETR101 gene expression, mainly through a CRE sequence via the CREB transcription pathway.

Animals↗
PubMed1989

Structure, sequence, and position of the stem-loop in tar determine transcriptional elongation by tat through the HIV-1 long terminal repeat.

The human immunodeficiency virus (HIV-1)-encoded trans-activator (tat) increases HIV gene expression and replication. Previously, we demonstrated that tat facilitates elongation of transcription through the HIV-1 long terminal repeat (LTR) and that short transcripts corresponding to prematurely terminated RNA are released and accumulate in the absence of tat. Here, using a transient expression assay, we tested clustered and compensatory mutations, as well as 3' deletions, in the trans-acting responsive region (tar) and observed that the primary sequence in the loop and secondary structure in the stem of the stem-loop in tar are required for trans-activation by tat. Insertions in the 5' region of tar revealed that tar must be near the site of HIV-1 initiation of transcription for trans-activation by tat. Deletions (3') and an insertion in tar demonstrated that an intact stem-loop is required for the recovery of prematurely terminated transcripts. Short and full-length transcripts were observed also with HIV type 2 (HIV-2) in the absence and presence of tat, respectively. We conclude that an intact stem-loop in tar is essential for trans-activation by tat and that initiation of transcription by HIV-1 promoter factors and elongation of transcription by tat are coupled.

Chloramphenicol O-Acetyltransferase↗
PubMed1994

Plant homeodomain protein involved in transcriptional regulation of a pathogen defense-related gene.

Transcription of the parsley pr2 gene, encoding pathogenesis-related protein 2 (PR2), is rapidly stimulated by fungal or bacterial elicitors. Previous work has revealed a 125-bp region within the pr2 promoter; this region encompasses all important cis-regulatory elements required for fungal elicitor-mediated expression. We now report the identification of a functionally relevant 11-bp DNA motif (CTAATTGTTTA) contained within this region; it specifically binds to factors present in both parsley and Arabidopsis nuclear protein extracts. From both plant species, full-length cDNA clones were isolated that encode proteins with high affinity fo this DNA motif. The proteins from both species contain stretches of 61 amino acids that are characteristic of homeodomain (HD) proteins. Binding studies and use of a polyclonal antiserum raised against a fusion polypeptide of glutathione S-transferase with the HD portion of the parsley protein indicated that the 11-bp DNA motif is a potential in vivo target site and that the HD protein is contained within the observed complex formed between the DNA motif and nuclear protein extracts. Transient expression studies using the authentic and a mutated target site suggested a functional role of the HD-DNA interaction in the regulation of the pr2 gene expression.

Amino Acid Sequence↗
PubMed2002

Clinical Features of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary disease characterized by recurrent transient ischemic attacks, strokes, and vascular dementia. Various mutations in the Notch3 gene cause the disease, whereas the mechanism of how they cause the disorder remains unknown. We recently identified two Japanese CADASIL families with an R141C mutation. The mean age of onset was 44.6, and the main symptoms were recurrent strokes and progressive motor disturbances in extremities, as well as pseudobulbar palsy. Besides those in white matter and basal ganglia, ischemic lesions in temporal edge and corpus callosum were present on magnetic resonance images, which seemed to be characteristic of CADASIL. Moreover, in our cases, nocturnal arterial blood pressure fall was significantly lower in patients compared with control subjects, suggesting that it might be partly associated with ischemic lesions in deep white matter in CADASIL. We also compared Japanese and Caucasian CADASIL cases and found that dementia and pseudobulbar palsy were observed more frequently in Japanese patients, although typical migraine was rather rare. In the present study, we describe the clinical features of CADASIL, hoping to help reveal the mechanism of chronic ischemic brain diseases, including leukoaraiosis or Binswanger's disease.

Adult↗
PubMed1992

Initial hydrogenation during catabolism of picric acid by Rhodococcus erythropolis HL 24-2.

Rhodococcus erythropolis HL 24-2, which was originally isolated as a 2,4-dinitrophenol-degrading bacterium, could also utilize picric acid as a nitrogen source after spontaneous mutation. During growth, the mutant HL PM-1 transiently accumulated an orange-red metabolite, which was identified as a hydride-Meisenheimer complex of picric acid. This complex was formed as the initial metabolite and further converted with concomitant liberation of nitrite. 2,4,6-Trinitrocyclohexanone was identified as a dead-end metabolite of the degradation of picric acid, indicating the addition of two hydride ions to picric acid.

Biodegradation, Environmental↗
PubMed1994

The transcriptional regulator YY1 binds to the 5'-terminal region of B19 parvovirus and regulates P6 promoter activity.

We performed a systematic study to identify cellular factors that bound to the terminal repeat region of B19 parvovirus. Using electrophoretic mobility shift assays, we detected one cellular factor which prominently bound to the repeat region. The factor was purified from K562 nuclear extract by specific DNA affinity column chromatography and identified as YY1, a multifunctional transcription factor. Of multiple possible YY1 binding sites in the upstream region of the P6 promoter, three showed specific strong binding. Transcription enhancement by YY1 was demonstrated in vitro by transient transfection assays. In studies using truncated and mutated versions of this region, YY1 activity was diminished by the alteration of any two of these three sites and abolished by the alteration of all three sites. Our results suggest that YY1 is a positive regulator of B19 parvovirus transcription.

Base Sequence↗
PubMed1996

Transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the RelA transcription factor as a mechanism producing airway mucosal inflammation.

The most common cause of epidemic pediatric respiratory disease, respiratory syncytial virus (RSV), stimulates interleukin-8 (IL-8) synthesis upon infecting airway epithelium, an event necessary for the development of mucosal inflammation. We investigated the mechanism for enhanced IL-8 production in human A549 type II pulmonary epithelial cells. Infection with sucrose-purified RSV (pRSV) produced a time-dependent increase in the transcriptional initiation rate of the IL-8 gene. Transient transfection of the human IL-8 promoter mutated in the binding site for nuclear factor-kappaB (NF-kappaB) demonstrated that this sequence was essential for pRSV-activated transcription. Gel mobility shift assays demonstrated pRSV induction of sequence-specific binding complexes; these complexes were supershifted only by antibodies directed to the potent NF-kappaB transactivating subunit RelA. Both Western immunoblot and indirect immunofluorescence assays showed that cytoplasmic RelA in uninfected cells became localized to the nucleus after pRSV infection. RelA activation requires replicating RSV, because neither conditioned medium nor UV-inactivated pRSV was able to stimulate its translocation. We conclude that RelA undergoes changes in subcellular distribution in airway epithelial cells upon pRSV infection. The ability of replicating RSV to activate RelA translocation may play an important role in activating IL-8 and other inflammatory gene products necessary for airway mucosal inflammation seen in RSV disease.

Cell Nucleus↗
PubMed1997

The N-terminal region of hepatitis C virus-encoded NS5A is important for NS4A-dependent phosphorylation.

We previously showed that two proteins, a 56-kDa protein (p56) and a 58-kDa protein (p58), are produced from the hepatitis C virus (HCV) nonstructural 5A region (NS5A) and that the production of p58 is enhanced by the presence of NS4A (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320-326, 1994). Both proteins have phosphorylated serine residues, some of which are located in the C-terminal region. In p58, phosphorylation of serine residues in the central region of HCV NS5A is important for production of p58 in an NS4A-dependent manner. To clarify the mechanism of NS5A phosphorylation, in particular phosphorylation in the central region, phosphorylation of deleted and mutated forms of NS5A was analyzed using a transient protein production system in cultured cells in the presence or absence of NS4A. Association of the NS5A region from amino acids 2135 to 2139 with NS4A was important for NS4A-dependent phosphorylation of NS5A.

Animals↗
PubMed1994

The basic helix-loop-helix protein upstream stimulating factor regulates the cardiac ventricular myosin light-chain 2 gene via independent cis regulatory elements.

Previous studies have documented that 250 bp of the rat cardiac ventricular myosin light-chain 2 (MLC-2v) promoter is sufficient to confer cardiac muscle-specific expression on a luciferase reporter gene in both transgenic mice and primary cultured neonatal rat myocardial cells. Utilizing ligation-mediated PCR to perform in vivo dimethyl sulfate footprinting, the present study has identified protein-DNA interaction within the position from -176 to -165. This region, identified as MLE1, contains a core sequence, CACGTG, which conforms to the consensus E-box site and is identical to the upstream stimulating factor (USF)-binding site of the adenovirus major late promoter. Transient assays of luciferase reporter genes containing point mutations of the site demonstrate the importance of this cis regulatory element in the transcriptional activation of this cardiac muscle gene in ventricular muscle cells. The protein complex that occupies this site is capable of binding to HF-1a and PRE B sites which are known to be required for cardiac muscle-specific expression of rat MLC-2v and alpha-myosin heavy-chain genes, respectively. This study provides direct evidence that USF, a member of the basic helix-loop-helix leucine zipper family, binds to MLE1, HF-1a, and PRE B sites and suggests that it is a component of protein complexes that may coordinately control the expression of MLC-2v and alpha-myosin heavy-chain genes. The current study also provides evidence that USF can positively and negatively regulate the MLC-2v gene via independent cis regulatory elements.

Animals↗
PubMed1998

Activation of Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein.

The small GTPase RhoA plays a critical role in signaling pathways activated by serum-derived factors, such as lysophosphatidic acid (LPA), including the formation of stress fibers in fibroblasts and neurite retraction and rounding of soma in neuronal cells. Previously, we have shown that ectopic expression of v-Crk, an SH2/SH3 domain-containing adapter proteins, in PC12 cells potentiates nerve growth factor (NGF)-induced neurite outgrowth and promotes the survival of cells when NGF is withdrawn. In the present study we show that, when cultured in 15% serum or lysophosphatidic acid-containing medium, the majority of v-Crk-expressing PC12 cells (v-CrkPC12 cells) display a flattened phenotype with broad lamellipodia and are refractory to NGF-induced neurite outgrowth unless serum is withdrawn. v-Crk-mediated cell flattening is inhibited by treatment of cells with C3 toxin or by mutation in the Crk SH2 or SH3 domain. Transient cotransfection of 293T cells with expression plasmids for p160ROCK (Rho-associated coiled-coil-containing kinase) and v-Crk, but not SH2 or SH3 mutants of v-Crk, results in hyperactivation of p160ROCK. Moreover, the level of phosphatidylinositol-4,5-bisphosphate is increased in v-CrkPC12 cells compared to the levels in mutant v-Crk-expressing cells or wild-type cells, consistent with PI(4)P5 kinase being a downstream target for Rho. Expression of v-Crk in PC12 cells does not result in activation of Rac- or Cdc42-dependent kinases PAK and S6 kinase, demonstrating specificity for Rho. In contrast to native PC12 cells, in which focal adhesions and actin stress fibers are not observed, immunohistochemical analysis of v-CrkPC12 cells reveals focal adhesion complexes which are formed at the periphery of the cell and are connected to actin cables. The formation of focal adhesions correlates with a concomitant upregulation in the expression of focal adhesion proteins FAK, paxillin, alpha3-integrin, and a higher-molecular-weight form of beta1-integrin. Our results indicate that v-Crk activates the Rho-signaling pathway and serves as a scaffolding protein during the assembly of focal adhesions in PC12 cells.

Animals↗
PubMed1987

Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.

We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.

Cell Line↗
PubMed2004

Insights into the molecular nature of magnesium homeostasis.

Magnesium is an important cofactor for many biological processes, such as protein synthesis, nucleic acid stability, or neuromuscular excitability. Extracellular magnesium concentration is tightly regulated by the extent of intestinal absorption and renal excretion. Despite the critical role of magnesium handling, the exact mechanisms mediating transepithelial transport remained obscure. In the past few years, the genetic disclosure of inborn errors of magnesium handling revealed several new proteins along with already known molecules unexpectedly involved in renal epithelial magnesium transport, e.g., paracellin-1, a key player in paracellular magnesium and calcium reabsorption in the thick ascending limb or the gamma-subunit of the Na(+)-K(+)-ATPase in the distal convoluted tubule. In this review, we focus on TRPM6, an ion channel of the "transient receptor potential (TRP) gene family, which, when mutated, causes a combined defect of intestinal magnesium absorption and renal magnesium conservation as observed in primary hypomagnesemia with secondary hypocalcemia.

Animals↗
PubMed2006

Role of the mitogen-activated protein kinase signaling pathway in the regulation of human melanocytic antigen expression.

Heterogeneous expression of melanocytic antigens occurs frequently in melanomas and represents a potent barrier to immunotherapy. We previously showed that coordinated losses of several melanocytic antigens are generally attributable to down-regulation of antigen gene expression rather than irreversible mutation. Treatment of melanoma cells with mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors blocks ERK activation and increases steady-state levels of mRNAs and corresponding protein expression for the melanocytic antigens Melan-A/MART-1, gp100, and tyrosinase. Although the degree of MEK inhibitor enhancement of antigen expression varied among different cell lines irrespective of their antigen expression status, all showed detectable responses. Notably, the antigen-enhancing effects of the MEK inhibitors could not be attributed to the master melanocytic regulator MITF-M. Because MAPK pathway activation via constitutively active mutant forms of BRAF is common in melanomas, correlation between BRAF function and antigen expression was investigated. No simple correlation of endogenous BRAF mutational status and antigen levels was observed, but transient overexpression of V600E BRAF increased ERK activation and reduced Melan-A/MART-1 levels in antigen-positive cell lines. These data indicate that whereas multiple factors may regulate antigen expression in melanomas, enhancement of MAPK signaling can act as a negative influence. Blocking such signaling with MEK inhibitors accordingly augments antigen levels, thereby enhancing Melan-A/MART-1-specific cytotoxic T-cell responses to antigen-negative cells following MEK inhibition treatment. Consequently, MAPK inhibition may assist targeting of melanomas for immunotherapy.

Antigens, Neoplasm↗
PubMed2003

Two novel mutations in the human coagulation factor VII promoter.

The factor VII genes of five unrelated Finnish female patients, F1-F5, with moderate bleeding tendency, were screened for mutations using single strand conformational polymorphisms and DNA sequencing. Heterozygous shifts were detected in exons 5 and 8 for patient F1, and sequencing confirmed the presence of the silent dimorphism H115H, the polymorphism R353Q and the mutation A294V. The patient F1 was also heterozygous for a novel -59T/G transversion mutation in the Hepatocyte nuclear factor 4-binding site. The remaining four patients carried a -32A/C transversion mutation located in a footprint (-51 to -32) covering the major transcription initiation start site -51). There was also a consensus sequence match to an initiator response-like binding element covering -51. Two patients were homozygous and two heterozygous for this mutation. Plasma FVII:Ag and FVII:C levels were reduced in parallel. A strong reduction in binding affinity of a specific nuclear protein to the -32C-containing oligonucleotide was found by electrophoretic mobility shift assays on nuclear extracts from HepG2 cells. EDTA caused no reduced binding. A minimal promoter (-191 to +15) containing the wild-type sequence or the -32A/C or -59T/G mutations was cloned in front of the firefly luciferase reporter gene and transiently transfected into Hep3B cells. Reduced activities [23.0 +/- 3.1% (-32C), 55.4 +/- 6.3% (-59G), 100% (wild-type construct)] were found for the mutated promoters. Southwestern blotting and UV crosslinking analysis showed binding of three proteins (20, 20 and 50 kDa) to the putative initiator response element. The -32A/C mutant oligonucleotide bound two proteins.

Adult↗
PubMed1998

Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit.

A novel in vivo experimental strategy, involving cell type-specific expression of a dominant-negative K+ channel pore-forming alpha subunit, was developed and exploited to probe the molecular identity of the cardiac transient outward K+ current (I(to)). A point mutation (W to F) was introduced at position 362 in the pore region of Kv4.2 to produce a nonconducting mutant (Kv4.2W362F) subunit. Coexpression of Kv4.2W362F with Kv4.2 (or Kv4.3) attenuates the wild-type currents, and the effect is subfamily specific; ie, Kv4.2W362F does not affect heterologously expressed Kv1.4 currents. With the use of the alpha-myosin heavy chain promoter to direct cardiac-specific expression, several lines of Kv4.2W362F transgenic mice were generated. Electrophysiological recordings reveal that I(to) is selectively eliminated in ventricular myocytes isolated from transgenic mice expressing Kv4.2W362F, thereby demonstrating directly that the Kv 4 subfamily underlies I(to) in the mammalian heart. Functional knockout of I(to) leads to marked increases in action potential durations in ventricular myocytes and to prolongation of the QT interval in surface ECG recordings. In addition, a novel rapidly activating and inactivating K+ current, which is not detectable in myocytes from nontransgenic littermates, is evident in Kv4.2W362F-expressing ventricular cells. Importantly, these results demonstrate that electrical remodeling occurs in the heart when the expression of endogenous K- channels is altered.

Action Potentials↗
PubMed2003

Diffuse neuroaxonal involvement in mucolipidosis IV as assessed by proton magnetic resonance spectroscopic imaging.

Mucolipidosis IV is an autosomal recessive disorder caused by mutations in MCOLN1, which codes for mucolipin, a transient receptor potential protein. In order to investigate brain metabolic abnormalities in mucolipidosis IV, we studied 14 patients (11 children, 3 adults) by proton magnetic resonance spectroscopic imaging. The ratios of N-acetylaspartate/ creatine-phosphocreatine and N-acetylaspartate/choline-containing compounds in patients with mucolipidosis IV were significantly reduced in all regions of interest except the parietal gray matter and thalamus. The ratios of choline-containing compounds/creatine-phosphocreatine was not significantly reduced in patients compared with controls. The ratio of N-acetylaspartate/creatine-phosphocreatine were significantly lower (P = .005) in the more neurologically impaired patients compared with the least impaired. For every region of interest, except for parietal gray matter, the ratio of N-acetylaspartate/creatine-phosphocreatine was lower in the more motorically impaired patient group. There was no difference for the ratio of N-acetylaspartate/creatine-phosphocreatine between younger and older patients. These findings suggest that mucolipidosis IV is largely a static developmental encephalopathy associated with diffuse neuronal and axonal damage or dysfunction. Mucolipin deficiency impairs motor more than sensory central nervous system pathways.

Adolescent↗