Biomedical subjects
Publications and source records attributed to N H Lee.
Two new 5-oxonoraporphines, 1 and 2, together with three known compounds, ouregidione, 3-methoxycepharadione B, and isoelemicin, have been isolated from the bark of Mitrephora cf. maingayi. Structures of 1 and 2 were determined to be 1,2,3-trimethoxy-5-oxonoraporphine and 1,2-dimethoxy-3-hydroxy-5-oxonoraporphine on the basis of NMR and MS studies.
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Pre-Sézary syndrome is an erythroderma with a chronic course, clinical findings of Sézary syndrome, lymphocytic subepidermal band infiltration at times, and repeated cycles of circulating Sézary cells of less than 1,000 cells/mm3. Duration of the pre-existing skin diseases preceding pre-Sézary erythroderma varies from a few weeks to 20 years. Before the erythroderma develops, these patients are diagnosed with contact dermatitis, neurodermatitis, chronic dermatitis, atopic dermatitis, or asteatotic eczema. Hand lesion also precedes the pre-Sézary erythroderma. This condition has been controlled by three cycles of chemotherapy consisting of vincristine, cytoxan, doxorubicin, and prednisolone. We describe a case of pre-Sézary syndrome preceded by hand lesion and treated with chemotherapy.
Nerve growth factor (NGF) induces differentiation of the rat pheochromocytoma clone (PC12) by activating the high affinity receptor, p140(trkA), linked to mitogen-activated protein kinase. While the physiological role of the low affinity NGF receptor (p75) has not been clearly defined, this receptor promotes activation of nuclear factor (NF) kappaB in Schwann cells. PC12 cells express the A(2A) adenosine receptor (AR), whose expression is significantly decreased by NGF treatment. In this study, we determined whether TrkA or p75 is involved in NGF-mediated regulation of A(2A)AR expression. NGF treatment decreased A(2A)AR in a time-dependent manner, with maximal effects observed by 1 day, and continued down-regulation of the receptor for up to 3 days in the presence of NGF. The decrease in A(2A)AR was associated with a more delayed decrease in the steady-state levels of the A(2A)AR mRNA. Down-regulation of the A(2A)AR at 1 day was mimicked by activators of NFkappaB, such as H(2)O(2), and ceramide, and was attenuated by the inhibitor pyrrolidine dithiocarbamate or following transient transfection of PC12 cells with a dominant negative IkappaBalpha mutant. Moreover, NGF stimulated nuclear accumulation of p65 subunits of NFkappaB (but not p50 subunits) in PC12 cells, as determined by electrophoretic mobility shift assays and by Western blotting. In contrast, inhibition of TrkA by AG879 or of TrkA-dependent mitogen-activated protein kinase mitogen-activated protein kinase kinase with PD98059 blocked PC12 cell differentiation without affecting A(2A)AR down-regulation, suggesting dissociation between these two phenomena. Taken together, these data provide strong support for the involvement of the p75/NFkappaB pathway in NGF-mediated down-regulation of A(2A)AR in PC12 cells.
Nerve growth factor (NGF) up-regulated steady-state levels of m4 muscarinic acetylcholine receptor (mAChR) mRNA in PC12 cells. Up-regulation of mRNA levels was associated with a corresponding increase in mAChR binding sites. Two other growth factors, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), up-regulated m4 mRNA and mAChR binding sites. Treatment of PC12 cells with NGF and bFGF, but not EGF, has previously been demonstrated to result in sustained activation of mitogen-activated protein kinase (MAPK). Analogously, NGF and bFGF, but not EGF, increased the stability of m4 mRNA in PC12 cells. In HER-PC12 cells, a clonal PC12 cell transfectant overexpressing EGF receptors and displaying sustained MAPK activation upon receptor stimulation, EGF treatment stabilized the m4 transcript. A synthetic inhibitor of MAPK kinase, PD98059, inhibited growth factor-induced stabilization of the m4 transcript in both PC12 and HER-PC12 cells. These findings demonstrate that the MAPK pathway is involved in transcript stabilization. Cycloheximide pretreatment abolished the post-transcriptional effect of NGF, indicating that de novo protein synthesis was required for the observed increase in m4 mRNA stability. By contrast, cycloheximide had no discernible post-transcriptional effect if added after NGF treatment, suggesting that an inducible yet stable protein factor was involved in m4 mRNA decay. An unusually well conserved 137 nucleotides of m4 3'-untranslated region has been identified by sequence comparison with other mRNAs that are post-transcriptionally regulated by NGF. In PC12 cells that heterologously overexpress this region, we demonstrate that NGF no longer stabilizes endogenous m4 mRNA. This conserved region probably represents an NGF-responsive element involved in mRNA stability regulation. Finally, transcription of the m4 gene can be induced by all three growth factors but is not dependent on MAPK activity, unlike growth factor-induced m4 mRNA stabilization.
Viral infections increase vagally mediated reflex bronchoconstriction. Decreased function of inhibitory M2 muscarinic receptors on the parasympathetic nerve endings is likely to contribute to increased acetylcholine release. In this study, we used cultured airway parasympathetic neurons to determine the effects of parainfluenza virus and of interferon (IFN)-gamma on acetylcholine release, inhibitory M2 receptor function, and M2 receptor gene expression. In control cultures, electrically stimulated acetylcholine release increased when the inhibitory M2 receptors were blocked using atropine (10(-)5 M) and decreased when these receptors were stimulated using methacholine (10(-)5 M). Acetylcholine release was increased by viral infection and by treatment with IFN-gamma (300 U/ml). In these cells, atropine did not further potentiate, nor did methacholine inhibit, acetylcholine release, suggesting decreased inhibitory M2 receptor function and/or expression. Using a competitive reverse transcription-polymerase chain reaction method, we demonstrated that M2 receptor gene expression was decreased by more that an order of magnitude both by virus infection and by treatment with IFN. Thus, viral infections may increase vagally mediated bronchoconstriction both by directly inhibiting M2 receptor gene expression and by causing release of IFN-gamma which inhibits M2 receptor gene expression.
BACKGROUND: Hepatocellular carcinoma (HCC) over 10 cm in diameter at the time of diagnosis continues to account for a number of patients undergoing hepatic resection. This study evaluated the clinicopathological features and outcome following surgery for large HCC. METHODS: Forty patients with a large HCC (greater than 10 cm) (group 1) resected between 1991 and 1996 were studied retrospectively. They were compared with 245 patients who had smaller HCCs (10 cm or less) (group 2). RESULTS: No patient in group 1 had hepatitis C infection compared with 22.9 per cent in group 2 (P=0.001). Patients in group 1 were significantly younger, had higher alpha-fetoprotein levels (16750 versus 1864 ng/ml; P < 0.001), better liver function, a higher incidence of multiple tumours (27 of 40 versus 42.0 per cent; P=0.003) and venous invasion (35 of 40 versus 52.2 per cent; P < 0.001), and underwent more major resections (37 of 40 versus 26.5 per cent; P < 0.001) than those in group 2. Morbidity and mortality rates and hospital stay were comparable in the two groups. For group 1, the 1-, 3- and 5-year disease-free survival rates were 42, 30 and 28 per cent respectively. Multiple tumours, venous invasion and impaired liver function were factors associated with recurrence. CONCLUSION: Large HCC had specific clinicopathological features. In selected patients, resection is safe and offers the chance of long-term disease-free survival.
Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.
We examined changes in expression of genes coding for alpha 2-AR subtypes in the dorsal root ganglion (DRG) in a rat model (spinal nerve ligation) or neuropathic pain. The present study demonstrates that the majority of DRG neurons express alpha 2C-AR mRNA and a small proportion of neurons express alpha 2A-AR mRNA, while few neurons express alpha 2B-AR mRNA in non-operated animals. In addition, alpha 2C- and alpha 2A mRNA levels in the DRG showed a significant decrease and increase, respectively in ligated animals. These findings suggest that alpha 2A- and alpha 2C-ARs in the DRG may play an important role in generating sympathetically maintained neuropathic pain.
Alanine scanning mutagenesis of B-B-X-X-B motifis (where B is a basic residue and X is any nonbasic residue) in m1 muscarinic acetylcholine receptors was performed to determine the relative roles of basic amino acids in receptor coupling. This conserved motif is found in many G protein-coupled receptors and has been implicated in G protein activation. The KKAAR365 motif, located at the carboxyl-terminal third intracellular loop of m1 receptors, was mutated to AAAAA365, thereby generating a triple-substitution mutant devoid of ability to stimulate either phosphoinositide (PI) hydrolysis or cAMP accumulation. In contrast, a triple-alanine substitution of the KRTPR140 motif in the carboxyl-terminal second intracellular loop, yielding mutant AATPA140, had no effect on receptor coupling to the two independent second messenger pathways. Analysis of a series of single- and double-substitution mutants demonstrate that all three basic residues of the KKAAR365 motif participate in efficient m1 receptor coupling. The presence of second and third basic residues in this motif was absolutely critical for full agonist recognition of a high and low affinity state of the receptor. Mutation of either Lys362 or Lys365, but not-Lys361, abolished guanine nucleotide-dependent conversion of agonist affinity states and correlated with an inability of full agonists to fully activate PI hydrolysis. The different combinatorial double-substitution mutants also revealed that Lys365 was necessary but not sufficient, in the context of the KKAAR365 motif, for efficient receptor coupling. This residue cannot facilitate full agonist-stimulated Pl hydrolysis in the absence of both Lys361 and Lys362. In comparison, the critical residue Lys362 was both necessary and sufficient. Substitution of nearby basic residues Lys361 and Lys365 with alanine yielded mutant AKAAA365, which exhibited partial ability to couple PI hydrolysis after full agonist stimulation. Therefore, Lys365 seems to function in a hierarchal (interdependent) manner with nearby basic residues, whereas Lys361 and Lys362 can act independent of surrounding basic residues to facilitate partial m1 receptor coupling after full agonist stimulation. In contrast, all three residues must be present for stimulation of PI hydrolysis by a partial agonist.
In an effort to identify new genes and analyse their expression patterns, 174,472 partial complementary DNA sequences (expressed sequence tags (ESTs)), totalling more than 52 million nucleotides of human DNA sequence, have been generated from 300 cDNA libraries constructed from 37 distinct organs and tissues. These ESTs have been combined with an additional 118,406 ESTs from the database dbEST, for a total of 83 million nucleotides, and treated as a shotgun sequence assembly project. The assembly process yielded 29,599 distinct tentative human consensus (THC) sequences and 58,384 non-overlapping ESTs. Of these 87,983 distinct sequences, 10,214 further characterize previously known genes based on statistically significant similarity to sequences in the available databases; the remainder identify previously unknown genes. Thirty tissues were sampled by over 1,000 ESTs each; only eight genes were matched by ESTs from all 30 tissues, and 227 genes were represented in 20 or more of the tissues sampled with more than 1,000 ESTs. Approximately 40% of identified human genes appear to be associated with basic energy metabolism, cell structure, homeostasis and cell division, 22% with RNA and protein synthesis and processing, and 12% with cell signalling and communication.
Nerve growth factor-induced differentiation of adrenal chromaffin PC-12 cells to a neuronal phenotype involves alterations in gene expression and represents a model system to study neuronal differentiation. We have used the expressed-sequence-tag approach to identify approximately 600 differentially expressed mRNAs in untreated and nerve growth factor-treated PC-12 cells that encode proteins with diverse structural and biochemical functions. Many of these mRNAs encode proteins belonging to cellular pathways not previously known to be regulated by nerve growth factor. Comparative expressed-sequence-tag analysis provides a basis for surveying global changes in gene-expression patterns in response to biological signals at an unprecedented scale, is a powerful tool for identifying potential interactions between different cellular pathways, and allows the gene-expression profiles of individual genes belonging to a particular pathway to be followed.
Regulation of muscarinic acetylcholine receptor (mAChR) subtype mRNAs was investigated in the human neuroblastoma cell line IMR-32 and in transfected CHO cells. IMR-32 cells express both m1 and m3 subtypes of mAChR. Exposure of IMR-32 cells to the muscarinic agonist, carbamylcholine (CBC) leads to a time dependent down-regulation of mAChRs which was maximal by 9 hours. mAChR activation resulted in a differential regulation of mAChR subtype mRNAs. m1 mAChR mRNA was down-regulated following 12 hours of agonist treatment and was associated with a decreased stability of the receptor transcript. In contrast, the m3 mAChR mRNA was resistant to agonist treatment for up to 24 hours. Using transfected CHO cells, we identified sequence elements within the 3'-untranslated region (3'-UTR) of the m1 mAChR gene which dictate agonist-induced destabilization of the m1 mAChR mRNA. Removal of these sequences abolished the ability of chronic agonist exposure to destabilize m1 mAChR mRNA. These findings suggest that sequence specific differences between m1 and m3 mAChR subtypes, which both preferentially couple to hydrolysis of phosphoinositides, may be responsible for differences in the regulation of mAChR gene expression.
The effects of chronic agonist exposure on receptor number (down-regulation) have been shown, in part, to be due to effects on mRNA levels. Agonist-mediated effects on muscarinic acetylcholine receptor (mAChR) mRNA were investigated in Chinese hamster ovary (CHO) cells stably transfected with m1 mAChR gene constructs containing the open reading frame and a series of deletions of the flanking 3'-untranslated region (3'-UTR). Carbachol (CBC) down-regulated m1 mAChRs encoded by the construct m1C1, an m1 mAChR transcript containing the entire flanking 3'UTR (nucleotides 1526-2622), in a time-dependent fashion with maximal decreases occurring by 12 h. Steady-state levels of m1C1 mRNA declined in a parallel fashion beginning 6 h after CBC pretreatment. Similar findings were obtained with m1C2, a construct which is missing all but 261 bases of flanking 3'-UTR (nucleotides (nt) 1526-1786). Since the rate of mRNA degradation represents an important potential regulatory mechanism to control the level of gene expression, we investigated the effects of CBC treatment on m1C1 and m1C2 mRNA stability. The half-life of either transcript in untreated cells was approximately 14 h, whereas m1C1 and m1C2 transcript half-lives decreased to approximately 3 h in cells treated with CBC. Agonist-induced destabilization of m1C2 mRNA could be mimicked by phorbol esters in a concentration-dependent manner and blocked by the protein kinase inhibitor, H-7. In contrast, m1 mAChR mRNA constructs missing nt 1526-1786 of the 3'-UTR (m1C3 and m1C4) did not undergo agonist- or phorbol ester-induced destabilization. In the neuroblastoma cell line IMR-32, endogenous m1 mAChR mRNA was down-regulated and destabilized following CBC treatment. These results demonstrate that agonist-induced mRNA destabilization is a potential mechanism for regulating m1 mAChR levels. Furthermore, deletion studies identify a 261 base region of the 3'-UTR having the potential to form stable stem-loop structures which likely harbors element(s) responsible for message destabilization.
High-throughput automated sequencing has enabled researchers to examine large numbers of clones from a cDNA library as a measure of the steady-state levels of mRNA species. The past year has witnessed many new applications of this technique to allow the qualitative and quantitative comparison of the changes in transcript levels from multiple genes.
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The sites of ectopic pregnancies in women who underwent various types of reconstructive tubal surgery were analysed and compared to the controls. Overall, ectopic pregnancies subsequent to all types of tubal surgeries were more often implanted proximally compared to the control population.
Genes encoding the m1 muscarinic (m1 mAChR) and beta 2-adrenergic receptors (beta 2AR) were stably co-expressed into Chinese hamster ovary (CHO) cells to study receptor regulation and cross-talk. Persistent activation of the beta 2AR/adenylate cyclase pathway by isoproterenol leads to heterologous desensitization, internalization, and down-regulation of the m1 mAChR which is comparable, but smaller in magnitude, with that seen with persistent activation of the m1 mAChR by carbachol. This heterologous effect was mimicked by dibutyryl cAMP and forskolin and antagonized by the protein kinase A (PKA) inhibitor H-8. A potential consensus sequence for phosphorylation by PKA (Lys351-Arg-Lys-Thr354) exists on the third intracellular loop of the m1 mAChR, suggesting that receptor phosphorylation by PKA may be involved in heterologous regulation. The loss of m1 mAChRs induced by carbachol was not reversed by H-8, indicating that homologous regulation is not dependent on PKA. Recent evidence suggests that muscarinic agonist-mediated internalization of the m1 mAChR involves the third intracellular loop (i3) (Maeda, S., Lameh, J., Mallet, W. G., Philip, M., Ramachandran, J., and Sadee, W. (1990) FEBS Lett. 269, 386-388). Three deletion mutant receptors were constructed in which the majority, or small regions, of i3 were eliminated but the membrane proximal portions of the loop were left intact. Each of the mutants was co-expressed with the beta 2AR in CHO cells. A small region in i3 was identified which is crucial for carbachol- and isoproterenol-promoted internalization and down-regulation. This region contains a series of 6 serine residues within an 8-amino acid stretch. A similar domain has been identified in the carboxyl tail of the beta 2AR and has been proposed to participate in receptor internalization (Hausdorff, W. P., Campbell, P. T., Ostrowski, J., Yu, S. S., Caron, M. G., and Lefkowitz, R. J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2979-2983).