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A S Lee

Publications and source records attributed to A S Lee.

At least 19 recordsLinked to original sources
PubMed1995

Generation of a mammalian cell line deficient in glucose-regulated protein stress induction through targeted ribozyme driven by a stress-inducible promoter.

GRP94 is an endoplasmic reticulum (ER) localized glycoprotein with Ca2+ binding and protein chaperoning properties. Using a ribozyme driven by a stress-inducible promoter and targeted against grp94 mRNA, we have generated a cell line deficient in its ability to induce GRP94. The effect of the ribozyme is mediated by the cleavage of the grp94 message just downstream of the initiation codon, and not by an antisense effect, as determined by the level of intact grp94 mRNA. Unexpectedly, this cell line's ability to induce GRP78 is also impaired. Transient overexpression of recombinant human lysosomal hydrolase alpha-L-iduronidase in the ribozyme expressing cells indicates that the secretion ratio of this enzyme is reduced by about 6-fold. Additionally, the ribozyme expressing cells showed increased sensitivity to Ca2+ depletion from ER caused by either A23187 or thapsigargin, an ER-Ca(2+)-ATPase inhibitor, but not to tunicamycin. These combined results show that the induction of GRP94 may play important roles in ER to nuclear signaling, protein sorting and secretion, and specific protection against Ca2+ depletion stress.

Animals↗
PubMed1995

Use of the stress-inducible grp78/BiP promoter in targeting high level gene expression in fibrosarcoma in vivo.

Current advances in human gene therapy open up new frontiers for molecular therapies of cancer. However, one major limitation in cancer gene therapy is the lack of a general tumor-specific promoter which allows stringent and high level expression of the therapeutic reagent in malignantly transformed but not normal tissues. Hallmark features of solid tumors such as glucose deprivation, chronic anoxia, and acidic pH induce the glucose-regulated proteins, in particular, GRP78/BiP, a M(r) 78,000 endoplasmic reticulum-localized protein with chaperone and calcium-binding properties. We report here that a truncated rat grp78 promoter with most of the distal basal elements removed can be utilized as a potent internal promoter in a retroviral vector to drive high level expression of a reporter gene in a murine fibrosarcoma model system. The stress-inducible grp78 promoter offers a novel approach for gene delivery systems targeting transcription in tumorigenic cells.

3T3 Cells↗
PubMed1995

Identification of a 68 kDa protein species as a specific DNA-binding component of the H3abp complex interacting with the histone H3.2 G1/S regulatory domain.

The hamster histone H3.2 promoter contains a protein binding site (referred to as site X) required for G1/S transcriptional activation. We report here that nuclear extracts prepared from serum synchronized cells at various stages of the cell cycle show a biphasic increase in the H3.2 specific complex, H3abp, binding to site X. An increase in binding activity occurs as cells first enter the cell cycle and later at the G1/S border. The H3.2 specific binding activity is enhanced by Mg2+ and Ca2+ in vitro, but is inhibited by Zn2+. Site X resembles a Jun/AP-1 site, but previously it has been shown that the H3abp complex is immunologically distinct from the characterized AP-1 proteins. Here, we identify the size of the hamster nuclear protein(s) that bind specifically to the H3abp site by ultra-violet crosslinking and renaturation of specific protein bands following gel electrophoresis. In addition, we purify H3abp by affinity chromatography and show that the purified H3abp has a different methylation interference profile from AP-1. Our results indicate that a protein species around 68 kDa is the major DNA binding component of the H3abp complex and it binds specifically to the histone promoter site required for G1/S regulation.

Animals↗
PubMed1995

Requirement of tyrosine- and serine/threonine kinases in the transcriptional activation of the mammalian grp78/BiP promoter by thapsigargin.

Depletion of endoplasmic reticulum (ER) Ca2+ store by thapsigargin (Tg) in mammalian cells induces a set of ER protein genes known as the glucose-regulated proteins. Recently, IRE1p, a transmembrane protein postulated to have a serine/threonine kinase activity, has been identified as required for the induction of ER resident proteins genes in Saccharomyces cerevisiae. To investigate whether IRE1p can stimulate mammalian grp transcription, a stable Chinese hamster ovary cell line containing amplified copies of IRE1p has been created. The IRE1p expressing transfectants exhibited a modest (2-fold) enhancement of both the basal and Tg induced level of grp78 and grp94, two coordinately regulated grp genes. Using okadaic acid as a specific inhibitor for the endogenous serine/threonine protein phosphatase activities, a mild (2-fold) stimulative effect was observed for Tg induction of grp78 transcription. The okadaic acid potentiating effect requires a 50-base pair region in the vicinity of the grp78 TATA element. In contrast, the transcriptional activation of grp78 by Tg is almost totally eliminated by genistein, a tyrosine kinase inhibitor. The grp core, the C3 and C1 elements which are major Tg response elements of the rat grp78 promoter, are also major targets of the inhibitive effects of genistein.

3T3 Cells↗
PubMed1995

Dysfunctional attitudes in depressed and recovered depressed patients and their first-degree relatives.

A series of depressed probands and their first-degree relatives were categorized as follows: (a) currently depressed; (b) recovered depressed; and (c) never-ill relatives. Their scores on a subscaled version of the Dysfunctional Attitude Scale (the DAS-24) were compared. The DAS total scores mirrored previous findings in that the total scores for recovered individuals returned to normal or near-normal levels. However, the dependency subscale scores remained elevated for the recovered depressed group. It is proposed that these results support the conclusion that self-report measures of cognitive vulnerability should focus on specific rather than global effects, if progress is to be made in the search for true vulnerability factors.

Achievement↗
PubMed1995

Conformation-defective herpes simplex virus 1 glycoprotein B activates the promoter of the grp94 gene that codes for the 94-kD stress protein in the endoplasmic reticulum.

GRP94 is a major glycoprotein in the endoplasmic reticulum with calcium-binding properties. Recently, GRP94 has been shown to bind to unassembled forms of multimeric proteins and peptides. We report here that GRP94 forms a stable association with the mutated form of the herpes simplex type virus 1 (HSV-1) glycoprotein B, but not with the fully processed viral protein. Both the glycosylated and unglycosylated forms of GRP94 are capable of complexing with the mutated, conformation-defective viral glycoprotein. Cotransfection of expression vectors for gB and grp94 promoter fusion genes revealed that the grp94 promoter is strongly activated by the mutant form of gB. Analysis of the grp94 promoter mutants showed that two regions in the promoter, a highly conserved element referred to as grp core and the CCAAT element most proximal to the TATA element (C1), mediate the induction of grp94 by malfolded protein. We further determined that the grp94 core and C1 element bind to common as well distinct nuclear factors from grp78, a commonly coregulated gene. Through UV cross-linking, site competition, and immunocross-reactivity, we identified that the heteromeric CCAAT-binding protein (CBF) is one component of the grp94 C1 complex.

Animals↗
PubMed1995

Transduction of calcium stress through interaction of the human transcription factor CBF with the proximal CCAAT regulatory element of the grp78/BiP promoter.

Mammalian GRP78/BiP is a stress-inducible 78-kDa endoplasmic reticulum (ER) protein with molecular chaperone and calcium-binding properties. The transactivation of grp78 by the calcium ionophore A23187 provides a model system with which to study the signal transduction that allows mammalian cells to sense calcium depletion in intracellular stores and activate transcription of specific genes. Linker-scanning mutation analysis of the grp78 promoter reveals that the single most important regulatory element is C1, which contains a CCAAT motif most proximal to the TATA sequence. The C1 element is crucial for mediating the stimulatory effects by the upstream regulatory elements under normal and stress conditions. In this report, we establish that the heteromeric CCAAT-binding factor CBF is the major component of the C1-binding factor (C1F) in human cells. A GGAGG motif flanking the CCAAT sequence also contributes to high-affinity C1F/CBF binding. We show here that the binding of C1F in vitro is sensitive to the concentration of calcium ions. At high calcium ion concentrations, the C1F-binding activity is lower because of a higher dissociation rate. This binding characteristic correlates with the induction of grp78 transcription in response to the depletion of intracellular calcium stores. The strikingly similar behavior of C1F from nuclear extracts of control and A23187-treated cells further suggests that C1F itself does not undergo any major inherent changes after calium depletion stress. Rather, its binding property could be modulated by the immediate calcium ionic environment in stressed and nonstressed cells. On the basis of the in vitro and in vivo site occupancies of C1F and other stress-inducible changes of upstream regulatory complexes, we present a model to explain how C1F and other upstream factors can synergistically activate grp78 transcription in calcium-depleted cells.

Animals↗
PubMed1995

Loss of heterozygosity on chromosome 11 q in breast cancer.

AIMS: Chromosome 11q23 seems to be a site of frequent mutation in cancer. It also contains loci such as ataxia telangiectasia with possible importance in the pathogenesis of breast tumours. The short arm of chromosome 11 has been studied extensively in breast cancer, but the long arm, in particular the distal part, has been studied less frequently. Cytogenetic analysis has shown possible involvement of chromosome 11q in breast tumours. Chromosome transfer experiments have also implicated chromosome 11q in breast cancer. A high frequency of mutations might therefore be expected to occur on chromosome 11q in breast cancers. METHODS: Using restriction fragment analysis, the primary tumours of 41 patients with breast cancer were screened for mutations at five loci on chromosome 11q (D11Z1, INT2, (FGF3), DRD2, NCAM, and D11S29). RESULTS: Allelic loss occurred at a high frequency (59%) at D11S29. At NCAM, novel alleles were frequently seen on autoradiographs. Relatively low frequencies of mutation were detected at the other loci. Allelic loss at D11S29 was associated with the presence of lymph node metastases, but this may be a chance association. CONCLUSIONS: The frequency of allelic loss at the DS11S29 locus is high. The significance of novel alleles at NCAM and their relation to allelic loss at D11S29 are unclear. The results presented here do not permit fine mapping of a region of allelic loss, but suggest that the region of greatest loss lies distal to DRD2. The results provide further evidence for the importance of gene(s) near 11q23 in the pathogenesis of breast cancer, and of tumours in general.

Breast Neoplasms↗
PubMed1995

Tuberculosis of the knee in children.

We reviewed 33 children with tuberculosis of the knee treated during the period from 1979 to 1991. All were treated with triple chemotherapy, using rifampicin, isoniazid and pyrazinamide for nine months. No patient had a synovectomy; surgery was limited to open biopsy or salvage procedures such as posterior release and arthrodesis for late stages of the disease. The radiological appearance of the knee at presentation predicted the outcome. The 30 patients with stage-1 (normal) or stage-2 (osteomyelitic) disease had excellent or good results; the three with narrowed joint spaces in stage 3 or stage 4 (arthritic) had fair or poor results. Early active mobilisation, as against long-term immobilisation, seemed to have no effect on the outcome of stage-1 or stage-2 disease.

Antitubercular Agents↗
PubMed1994

Localization of the gene encoding human BiP/GRP78, the endoplasmic reticulum cognate of the HSP70 family, to chromosome 9q34.

BiP/GRP78 is a member of the HSP70 family involved in the folding and assembly of proteins in the endoplasmic reticulum. Using PCR amplification of DNA from human x rodent somatic hybrids that segregate human chromosomes in conjunction with fluorescence in situ hybridization, we have assigned GRP78 to chromosome 9q34. This is in agreement with the localization of murine and bovine homologues based on the high degree of synteny in this region. Several interesting genes and disorders map to this region and are discussed.

Base Sequence↗
PubMed1994

Partial sequence analysis of the hepatitis C viral genome in Singapore patients.

The distribution of hepatitis C viral (HCV) genotypes in Singapore has not been previously determined. We studied the sera of 40 Singapore patients which were PCR-positive for HCV. The HCV genotypes were determined by direct sequencing of amplified sequences of the 5' non-coding region, after reverse transcription. Of the 40 samples, 35/40 (87.5%) were of HCV type 1, 2/40 (5.0%) were of type 2 and 3/40 (7.5%) were of type 3. The most common HCV genotype in this study was the type 1 genotype. Our results confirm the wide geographical distribution of HCV genotypes.

Hepacivirus↗
PubMed1994

Erp61 is GRP58, a stress-inducible luminal endoplasmic reticulum protein, but is devoid of phosphatidylinositide-specific phospholipase C activity.

Using antibody raised against putative Form I phosphatidylinositide-specific phospholipase C (PI-PLC) and direct amino acid sequencing of the protein recognized by this antibody, we have shown that the antibody reacts with luminal endoplasmic reticulum (ER) proteins, including ERp61. ERp61 possesses a COOH-terminal QEDL sequence that acts as an ER retention signal. Additional experiments have shown, however, that PI-PLC activity is separable from ERp61 and that rat or murine ERp61 expressed in COS cells failed to produce an increase in PI-PLC activity in the COS cells. Finally, we have identified ERp61 as GRP58, a 58-kDa protein inducible by glycosylation block and treatment with the Ca2+ ionophore, A23187.

Amino Acid Sequence↗
PubMed1994

Increased cytotoxicity of chronic hypoxic cells by molecular inhibition of GRP78 induction.

PURPOSE: To develop a molecular strategy of increasing cytotoxicity of chronically hypoxic cells by inhibiting Glucose Regulated Protein 78 kDal (GRP78) induction. METHODS AND MATERIALS: A mutant nonGRP78 inducing cell line (78WO) was developed from its parent (DG44) by overexpressing antisense GRP78 mRNA. Following exposure to varying durations of hypoxia, Northern and Western blot analysis were used to characterize the amount of GRP78 expression both at the RNA and protein level. Hypoxia was achieved by placing cells in specially designed hypoxic chambers which were subjected to successive rounds of evacuation and flushing with 95% CO2/N2 to reduce the oxygen in the environment to 0.02% oxygen. After treatment with hypoxia, cells were assayed for colony forming ability. RESULTS: GRP78 mRNA and protein induction following exposure to hypoxia was 3-4 fold lower in the 78WO cell line than in the parental DG44 cell line. Furthermore, it was observed that there was no difference in the cytotoxicity of 78WO and DG44 cells after 10 h of hypoxia. However, after 15 h of hypoxia, the survival of 78WO cells decreased by 1 log and after 20 h of hypoxia, the survival of 78WO decreased by another log. CONCLUSION: These results show that stress protein induction is important for cellular survival to chronic hypoxia and that inhibition of GRP78 induction may represent a novel therapeutic strategy by selectively sensitizing chronically hypoxic cells within solid tumors.

Amino Acid Sequence↗
PubMed1994

A structural role for arginine in proteins: multiple hydrogen bonds to backbone carbonyl oxygens.

We propose that arginine side chains often play a previously unappreciated general structural role in the maintenance of tertiary structure in proteins, wherein the positively charged guanidinium group forms multiple hydrogen bonds to backbone carbonyl oxygens. Using as a criterion for a "structural" arginine one that forms 4 or more hydrogen bonds to 3 or more backbone carbonyl oxygens, we have used molecular graphics to locate arginines of interest in 4 proteins: Arg 180 in Thermus thermophilus manganese superoxide dismutase, Arg 254 in human carbonic anhydrase II, Arg 31 in Streptomyces rubiginosus xylose isomerase, and Arg 313 in Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase/oxygenase. Arg 180 helps to mold the active site channel of superoxide dismutase, whereas in each of the other enzymes the structural arginine is buried in the "mantle" (i.e., inside, but near the surface) of the protein interior well removed from the active site, where it makes 5 hydrogen bonds to 4 backbone carbonyl oxygens. Using a more relaxed criterion of 3 or more hydrogen bonds to 2 or more backbone carbonyl oxygens, arginines that play a potentially important structural role were found in yeast enolase, Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase, bacteriophage T4 and human lysozymes, Enteromorpha prolifera plastocyanin, HIV-1 protease, Trypanosoma brucei brucei and yeast triosephosphate isomerases, and Escherichia coli trp aporepressor (but not trp repressor or the trp repressor/operator complex).(ABSTRACT TRUNCATED AT 250 WORDS)

Aldose-Ketose Isomerases↗
PubMed1994

Constitutive expression of a microinjected glucose-regulated protein (grp78) fusion gene during early Xenopus laevis development.

In this study we have found that a rat glucose-regulated protein (grp) 78 chloramphenicol acetyltransferase (CAT) fusion gene deleted to -456 bp at the 5' end and injected into fertilized Xenopus eggs was first expressed in a constitutive manner in late blastula stage embryos and displayed increased expression as the embryos developed to the gastrula and neurula stages. Using a series of internal deletion mutants and linker-scanner mutants of the rat grp78 promoter, we have found that a CCAAT box and CCAAT-like element within the region -129 to -90 were essential for constitutive expression of the chimeric genes in neurula stage embryos. These results suggest conservation of the regulatory sequences within the grp78 promoter between rat and Xenopus. Interestingly, deletion or alteration of sequences between -130 and -149 had a dramatic stimulatory effect on basal promoter activity. This effect, which was not observed previously in rat cells, may be the result of upstream elements that are transcriptionally active in Xenopus and that can compensate for the mutated or deleted sequences. It is also possible that these results indicate the presence of a negative regulatory element that is recognized by the Xenopus transcriptional apparatus.

Animals↗
PubMed1994

Stress induction of the mammalian GRP78/BiP protein gene: in vivo genomic footprinting and identification of p70CORE from human nuclear extract as a DNA-binding component specific to the stress regulatory element.

GRP78, also known as BiP, is one of the better-characterized molecular chaperones. It has been implicated in protein folding and also calcium sequestration in the endoplasmic reticulum. When the cells are subjected to endoplasmic reticulum stress, in particular the depletion of stored calcium and/or the accumulation of abnormal proteins, the rate of transcription of grp78 is enhanced. Previous studies have shown that the core region of the rat grp78 promoter (-170 to -135), which is 95% conserved with the human grp78 core (-133 to -98), is one of the key regulatory elements. Using ligation-mediated PCR, we have found that there are specific changes in factor occupancy after stress induction and the major changes occur within a cluster of bases located in the 3' half of the grp core, whereas other regulatory elements are constitutively occupied. This inducible binding to the 3' half of the human grp78 core region is observed under diverse stress signals, suggesting a common mechanism for the grp stress response. Nonetheless, the lack of constitutive in vivo protection at this region is not due to the absence of a binding factor in nuclear extracts. Using in vitro gel mobility shift assays, we detected a constitutive binding activity which exhibits specificity and affinity to the stress-inducible region. Through sodium dodecyl sulfate-polyacrylamide gel electrophoresis size fractionation and renaturation analysis, the activity is found in polypeptides with molecular sizes of 65 to 75 kDa. After a three-step purification scheme including core affinity column chromatography, we purified p70CORE, which is about 70 kDa in its monomeric form. The purified p70CORE is sufficient to form a complex specific to the stress-inducible region.

Base Sequence↗
PubMed1994

ANA negative systemic lupus erythematosus.

It is usually assumed that psychiatric disturbance cannot be caused by SLE alone. We report two cases of ANA-negative SLE presenting with psychiatric features.

Antibodies, Antinuclear↗