Regulation of mouse hepatitis virus RNA synthesis by heterogeneous nuclear ribonucleoprotein A1.
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Animals↗
Biomedical subjects
H P Li
Publications and source records attributed to H P Li.
Food poisoning due to methamidophos-contaminated vegetables.
BACKGROUND: Organophosphate poisoning is well known for its characteristic symptoms and signs, but food poisoning caused by pesticide-contaminated food is seldom reported. CASE REPORT: We report three incidents of food poisoning that resulted from exposure to the organophosphate insecticide methamidophos in vegetables. These outbreaks caused a cholinergic syndrome in 4 patients. The cholinergic overactivity led as to suspect organophosphate food poisoning. All patients recovered well following appropriate therapy. The clinical diagnosis of organophosphate poisoning was confirmed by reduced levels of erythrocytes and plasma cholinesterase and the presence of methamidophos in the vegetable leftovers. The implicated vegetables and levels of methamidophos were: Ipomoea batatas 255 ppm, Gynura bicolor 110 ppm, and red cabbage 26.3 ppm. Since methamidophos is normally applied to vegetables during planting, improper selection and/or overuse of pesticide or improper harvest times may explain the occurrence of these high residue levels of methamidophos.
Adult↗
Fatal poisoning by butachlor and chlornitrofen ingested from a bottle marked as nitrofen.
Nitrofen has been banned in Taiwan since January 1, 1983 due to its tetrogenicity. A 78-y-o female consumed about 500 ml of herbicide, labeled as nitrofen, in a suicide attempt. Consciousness disturbance occurred immediately. After 9 h, bloody-tarry stool was noted. Hematemesis occurred 19 h later. Hepatotoxicity and nephrotoxicity also developed. Twitching of head and both upper extremities lasting 20-30 sec and up to 5 min developed on day 3. Sometimes an upward gaze with the face turned to the right or a convulsion of the right upper extremity was observed. EEG showed alpha coma, generalized slow spike-and-wave complexes, and isolated generalized sharp waves mainly in the left occipital area. On day 7, endotracheal intubation was performed due to apnea; she expired on day 9. The pesticide was found to contain butachlor and chlornitrofen. The Agricultural Committee prohibited the marketing of chlornitrofen due to its generation of tumors in animals. The combination of butachlor and chlornitrofen can result in consciousness disturbance, leucocytosis, gastrointestinal hemorrhage, rhabdomyolysis, hypocalcemia, hypoalbuminemia, elevated amylase, nephrotoxicity, hepatotoxicity, seizures and death. The cytotoxicity of butachlor may be related to the patient's death. Active components of pesticides should be checked if the clinical course of a poisoned patient is unusual to allow appropriate interventions.
Acetanilides↗
Modification of the composition of polycystin-1 multiprotein complexes by calcium and tyrosine phosphorylation.
Mutations in the PKD1 gene are responsible for >85% of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1, polycystin-1, is a large, modular membrane protein, with putative ligand-binding motifs in the extracelluar N-terminal portion, 9-11 transmembrane domains and an intracellular C-terminal portion with phosphorylation sites. A role for polycystin-1 as a cell surface receptor involved in cell-matrix and cell-cell interactions has been proposed. In this study, we have analyzed polycystin-1 and associated protein distribution in normal human epithelial cells and examined the role of cell-matrix versus cell-cell interactions in regulation of the assembly of polycystin-1 multiprotein complexes. Immunocytochemistry, sucrose density gradient sedimentation, co-immunoprecipitation analyses and in vitro binding assays have shown that polycystin-1 associates with the focal adhesion proteins talin, vinculin, p130Cas, FAK, alpha-actinin, paxillin and pp60c-src in subconfluent normal human fetal collecting tubule (HFCT) epithelia when cell-matrix interactions predominate. Polycystin-1 also forms higher S value complexes with the cell-cell adherens junction proteins E-cadherin, beta- and gamma-catenins in confluent cultures when cell-cell interactions are predominant. Polycystin-1 multiprotein complexes can be disrupted by cytochalasin D but not by colchicine, suggesting involvement of the actin cytoskeleton. Although inhibition of tyrosine phosphorylation by tyrphostin inhibits polycystin-1-FAK interactions, E-cadherin interactions are enhanced. High calcium treatment also increases polycystin-1-E-cadherin interactions.
Cadherins↗
Population structure and history in East Asia.
Archaeological, anatomical, linguistic, and genetic data have suggested that there is an old and significant boundary between the populations of north and south China. We use three human genetic marker systems and one human-carried virus to examine the north/south distinction. We find no support for a major north/south division in these markers; rather, the marker patterns suggest simple isolation by distance.
China↗
Heterogeneous nuclear ribonucleoprotein A1 regulates RNA synthesis of a cytoplasmic virus.
Heterogeneous nuclear ribonucleoprotein (hnRNP A1) is involved in pre-mRNA splicing in the nucleus and translational regulation in the cytoplasm. In the present study, we demonstrate that hnRNP A1 also participates in the transcription and replication of a cytoplasmic RNA virus, mouse hepatitis virus (MHV). Overexpression of hnRNP A1 accelerated the kinetics of viral RNA synthesis, whereas the expression in the cytoplasm of a dominant-negative hnRNP A1 mutant that lacks the nuclear transport domain significantly delayed it. The hnRNP A1 mutant caused a global inhibition of viral mRNA transcription and genomic replication, and also a preferential inhibition of the replication of defective-interfering RNAs. Similar to the wild-type hnRNP A1, the hnRNP A1 mutant complexed with an MHV polymerase gene product, the nucleocapsid protein and the viral RNA. However, in contrast to the wild-type hnRNP A1, the mutant protein failed to bind a 250 kDa cellular protein, suggesting that the recruitment of cellular proteins by hnRNP A1 is important for MHV RNA synthesis. Our findings establish the importance of cellular factors in viral RNA-dependent RNA synthesis.
Animals↗
Interlaboratory study of identification and quantitation of multiresidue pyrethroids in agricultural products by gas chromatography-mass spectrometry.
This paper deals with the different GC-MS analytical conditions adopted by four laboratories in an attempt to confirm the accuracy of the GC-electron-capture detection (ECD) analytical results during the international collaborative study for the establishment of the AOAC Official Method 998. 01. What is especially noted is that two laboratories have conducted comparative analysis of the respective 12 blind samples with both methods of GC-ECD and GC-MS, and the analytical results of the two methods turn out to be basically identical. This fully demonstrates that GC-MS is not only an effective confirmation tool in the analysis of the pyrethroid residues but also of sufficient sensitivity regarding the maximum residue limit of determination prescribed by FAO/WHO. Moreover, its selectivity is better than GC-ECD.
Gas Chromatography-Mass Spectrometry↗
Low genetic variation of the Yunnan hare (Lepus comus G. Allen 1927) as revealed by mitochondrial cytochrome b gene sequences.
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Animals↗
Formation of a ribonucleoprotein complex of mouse hepatitis virus involving heterogeneous nuclear ribonucleoprotein A1 and transcription-regulatory elements of viral RNA.
The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) specifically binds to two transcription-regulatory elements, i.e., the leader and intergenic sequence, of the negative-strand (template-strand) RNA of mouse hepatitis virus (MHV) and may play a role in viral RNA transcription. Previous studies based on the defective-interfering RNAs of MHV suggested that these two RNA elements may interact with each other during transcription, although they do not have complementary sequences. In this study, we showed by an in vitro reconstitution assay that hnRNP A1 could mediate the formation of an RNP complex involving these two RNA elements. Both the RNA-binding domains and protein-interacting domain of hnRNP A1 contributed to the efficient formation of the RNP complex; however, the presence of the two RNA-binding domains alone, without the protein-interacting domain, also resulted in some RNP formation. Omission of hnRNP A1 in the reconstitution reaction abolished the RNP formation, and mutations of the IG sequences significantly inhibited the RNP formation. These findings suggest that the two cis-acting transcription-regulatory sequences of MHV RNA can interact with each other through the formation of an RNP complex involving a cellular protein hnRNP A1. This RNP complex may participate in MHV RNA transcription.
Animals↗
Identification of phosphorylation sites in the PKD1-encoded protein C-terminal domain.
The PKD1-encoded protein, "polycystin-1", has a large N-terminal extracellular portion, multiple transmembrane domains, and a short intracellular C-terminal tail with four tyrosine residues and two putative sites for serine phosphorylation. Its function in kidney development and autosomal dominant polycystic kidney disease (ADPKD) is still unknown. We have subcloned the cDNA encoding the polycystin-1 C-terminal domain (PKD1-CTD) into a prokaryotic expression vector, and site-directed mutagenesis was performed to target the four tyrosine residues and four serine residues in two putative phosphorylation sites. In vitro phosphorylation assays were conducted on both wild type and mutant PKD1-CTD fusion proteins. It was found that the wild type PKD1-CTD and all mutant fusion proteins, except S4251G/S4252G, could be phosphorylated by lysates from cultured normal human renal collecting tubule (NHCT) cells, as well as by commercially purified cAMP-dependent protein kinase (PKA). The phosphorylation of the PKD1-CTD fusion protein by NHCT lysates was greatly enhanced by cAMP and its analog 8-Br-cAMP, and inhibited by the specific PKA inhibitors PKI(6-22) and H-89. Activators and inhibitors of protein kinase C (PKC) had no effects on the phosphorylation of the PKD1-CTD fusion protein. Using commercially purified pp60(c-src) (c-src) it was also shown that the PKD1-CTD fusion protein could be phosphorylated by c-src in vitro, and that this phosphorylation could be abolished by a mutation Y4237F. By comparing the amino acid sequence at 4249-4253 (RRSSR) with the consensus sequence for PKA phosphorylation (RRXSX), we suggest that the serine residue at 4252 is the target of phosphorylation by a cAMP-dependent protein kinase in NHCT cell lysates. In addition, we suggest that Y4237 might be phosphorylated by c-src in living cells.
Cells, Cultured↗
Polypyrimidine tract-binding protein binds to the leader RNA of mouse hepatitis virus and serves as a regulator of viral transcription.
A cellular protein, previously described as p55, binds specifically to the plus strand of the mouse hepatitis virus (MHV) leader RNA. We have purified this protein and determined by partial peptide sequencing that it is polypyrimidine tract-binding protein (PTB) (also known as heterogeneous nuclear ribonucleoprotein [hnRNP] I), a nuclear protein which shuttles between the nucleus and cytoplasm. PTB plays a role in the regulation of alternative splicing of pre-mRNAs in normal cells and translation of several viruses. By UV cross-linking and immunoprecipitation studies using cellular extracts and a recombinant PTB, we have established that PTB binds to the MHV plus-strand leader RNA specifically. Deletion analyses of the leader RNA mapped the PTB-binding site to the UCUAA pentanucleotide repeats. Using a defective-interfering RNA reporter system, we have further shown that the PTB-binding site in the leader RNA is critical for MHV RNA synthesis. This and our previous study (H.-P. Li, X. Zhang, R. Duncan, L. Comai, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 94:9544-9549, 1997) combined thus show that two cellular hnRNPs, PTB and hnRNP A1, bind to the transcription-regulatory sequences of MHV RNA and may participate in its transcription.
5' Untranslated Regions↗
Cellular protein hnRNP-A1 interacts with the 3'-end and the intergenic sequence of mouse hepatitis virus negative-strand RNA to form a ribonucleoprotein complex.
We previously showed that several cellular proteins specifically bind to the 3'-end and the intergenic sequences of the negative-strand RNA of mouse hepatitis virus (MHV), and proposed that these distant RNA sequences can be brought together by cellular and viral proteins (Furuya and Lai, 1993; Zhang et al., 1994; Zhang and Lai, 1995). The cellular protein p35 has been identified as a heterogeneous nuclear ribonucleoprotein (hnRNP) A1. We have now expressed hnRNP-A1 as a glutathione-S-transferase (GST) fusion protein and demonstrated that the amino terminal two-thirds of hnRNP-A1 interacted with the two MHV regulatory RNA sequences (3'-end and intergenic sequences) through protein-RNA interaction while its carboxy-terminal glycine-rich domain mediated homomeric (protein-protein) interactions. In a partially reconstituted reaction, in which the two MHV RNA fragments and the purified GST-hnRNP-A1 fusion protein were mixed, an RNP complex was formed. Depletion of either hnRNP-A1 or one of the RNA components abolished the complex formation. These results indicate that hnRNP-A1 can mediate the formation of an MHV RNP complex, which includes both the negative-strand leader and intergenic sequences. Site-directed mutagenesis revealed that mutations in the MHV intergenic sequences, which inhibited MHV RNA transcription, also inhibited the RNP complex formation. Deletion analysis showed that the amino terminal RNA-binding domains of hnRNP-A1 is essential for the RNP complex formation while the carboxy-terminal protein-binding domain enhanced the complex formation by 90-fold. These findings provide direct evidence demonstrating that the negative-strand leader RNA and intergenic sequences can form an RNP complex mediated by cellular protein hnRNP-A1.
Animals↗
Effect of chinonin on acute hypoxic pulmonary vasoconstriction in Sprague-Dawley rats.
The objective of this study was to determine the influence of chinonin on acute hypoxic pulmonary vasoconstriction (HPV) in Sprague-Dawley (SD) rats and investigate its mechanism. Sixty-five SD rats were divided into five groups at random: six in the control group; six in the hypoxia group; 13 in the group of hypoxia with chinonin; 20 in the group of endothelin-1 (ET-1) with chinonin and 20 in the group of platelet activating factor (PAF) with chinonin. Their mean pulmonary arterial pressures (mPAP) were measured and the concentration of molondialdehyde (MDA) in plasma and the activity of phospholipase A2 (PLA2) of the lung tissues were detected. The PAF and ET-1 levels of plasma and lung homogenates were detected in the control and hypoxia groups. There was evidence of an increase in mPAP, MDA, PAF and ET-1 in the plasma, and activity of PLA2, PAF and ET-1 of the lung tissues when the rats inhaled a 10% mixture of oxygen in nitrogen. It appeared that chinonin may have been inhibiting the action of ET-1 and PAF. Chinonin could have prevented an increase in MPAP caused by hypoxia and inhibited the action of ET-1 and PAF. But chinonin had no influence on the increase in MDA in the plasma and the PLA2 activity of the lungs when hypoxia occurred. Chinonin can reduce HPV, but does not influence normal mPAP. It may do this by blocking the action of ET-1 and/or PAF or others. The definite mechanism needs to be studied further.
Animals↗
Mouse hepatitis virus infection induces an early, transient calcium influx in mouse astrocytoma cells.
Mouse hepatitis virus (MHV), a murine coronavirus, utilizes murine carcinoembryonic antigens as receptors. The events that follow virus-receptor binding and eventually lead to virus entry are poorly understood. We studied the possible effects of MHV infection on intracellular calcium in a mouse astrocytoma cell line. Using the calcium-sensitive dye fluo-3 and confocal laser scanning microscopy, we found that MHV strain JHM induced an immediate (within 20 s) and transient (lasting no longer than 2 min) calcium increase in about 5% of the infected cells. The calcium increase was blocked by antibodies against the viral spike protein, suggesting that it was specifically triggered by the interaction of the viral spikes with cells. It was also inhibited by L-type calcium channel blockers and was not detected in calcium-free medium, suggesting that the calcium increase was caused by calcium influx from the extracellular medium. Studies of the kinetics of viral replication by immunofluorescence staining of the viral nucleocapsid protein revealed that at 3 h postinfection there was roughly the same percentage of cells (5%) that produced the viral protein as the percentage of cells that had responded with a calcium signal. This finding and the virus dilution studies together suggest that calcium responders may represent cells that had been infected with multiple viruses and undergone rapid viral replication. Furthermore, calcium channel blockers, including verapamil and cadmium chloride, and the calcium chelator EGTA inhibited virus infection. Therefore, the transient intracellular calcium increase reported here may be an early signaling event associated with virus infection.
Aniline Compounds↗
Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA.
A cellular protein, previously described as p35/38, binds to the complementary (-)-strand of the leader RNA and intergenic (IG) sequence of mouse hepatitis virus (MHV) RNA. The extent of the binding of this protein to IG sites correlates with the efficiency of the subgenomic mRNA transcription from that IG site, suggesting that it is a requisite transcription factor. We have purified this protein and determined by partial peptide sequencing that it is heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an abundant, primarily nuclear protein. hnRNP A1 shuttles between the nucleus and cytoplasm and plays a role in the regulation of alternative RNA splicing. The MHV(-)-strand leader and IG sequences conform to the consensus binding motifs of hnRNP A1. Recombinant hnRNP A1 bound to these two RNA regions in vitro in a sequence-specific manner. During MHV infection, hnRNP A1 relocalizes from the nucleus to the cytoplasm, where viral replication occurs. These data suggest that hnRNP A1 is a cellular factor that regulates the RNA-dependent RNA transcription of the virus.
Animals↗
Immunohistochemical localization of neurocan and L1 in the formation of thalamocortical pathway of developing rats.
We used immunohistochemistry to examine possible molecular interactions between the subplate and growing thalamocortical axons in rat fetuses. In the cortical anlage of embryonic day 16 (E16), the subplate first appeared below the cortical plate. Among chondroitin sulfate proteoglycans, phosphacan was uniformly distributed throughout the cortical wall, whereas neurocan was localized only in the subplate at E16. Neural cell adhesion molecules, NCAM-H, TAG-1, and L1, were detected in the cortical anlage. Both cortical neurons and growing axons were diffusely immunopositive for NCAM-H, and TAG-1 immunoreactivity was found on immature neurons and cortical efferent axons but not on thalamocortical axons. L1 immunoreactivity was specifically localized on the growing thalamocortical axons. When the locations of neurocan and L1 were compared in the developing cortex, L1-bearing axons were found to extend to neurocan-immunopositive regions; neurocan immunoreactivity was intense in the subplate at E16, when small numbers of L1-immunoreactive thalamocortical axons began to invade the cortex. At E17, many L1-positive axons were observed in the subplate that expressed neurocan specifically. Double immunostaining showed that L1-positive axons and neurocan immunoreactivity overlapped in the subplate at E17. After E18, neurocan expression gradually extended to the lower part of the cortical plate; it extended to the entire cortex by E21, 1 day before birth. By E21, L1-bearing axons had invaded the lower part of the cortical plate. The present study demonstrated that the neurocan expression precedes growth of L1-bearing thalamocortical afferent fibers. Because neurocan can bind to L1 molecule in vitro, these results suggest that neurocan and L1 play some important roles in pathfinding of the thalamocortical afferent fibers during rat corticogenesis.
Animals↗
Kinesin-73 in the nervous system of Drosophila embryos.
Kinesin-73 cDNA was shown to encode a kinesin heavy chain protein that contains an N-terminal motor domain and a long central region that lacks extensive coiled-coils. The amino acid sequence of the motor domain of kinesin-73 protein is most closely related to the motor domains of Caenorhabditis elegans unc-104 and mouse KIF1A. The central region of kinesin-73 protein also is related to unc-104 and KIF1A, but the homology is lower than that of the motor domain. The C-terminal region of kinesin-73 protein contains a cytoskeleton associated protein Gly-rich domain, which is a putative microtubule binding site that is present in some cytoskeleton or dynein-associated proteins. Kinesin-73 mRNA was shown by in situ hybridization to be maternally expressed and widely distributed in the syncytial blastoderm embryo. However, later in Drosophila embryo development, expression of the kinesin-73 gene becomes restricted mostly to the central and peripheral nervous systems.
Amino Acid Sequence↗
Midodrine for the treatment of intradialytic hypotension.
Recurrent intradialytic hypotension is probably the most severely disabling feature in dialysis patients and the etiology is multifactorial. We assessed the efficacy and safety of midodrine, a selective alpha1-adrenergic pressor agent in 12 patients with recurrent intradialytic hypotension. Symptomatic intradialytic hypotension was defined as hypotensive symptoms occurring with a systolic blood pressure < 100 mm Hg or with 25% decrease in systolic blood pressure in those patients with a basal systolic blood pressure of 100 mm Hg. The patients who suffered from symptomatic intradialytic hypotension and failed to improve after cautious body weight adjustment were included into the study. The lowest intradialytic and postdialysis blood pressures were monitored for 18 consecutive dialysis sessions before and after midodrine treatment. Clinical signs and symptoms were also recorded during both periods. With midodrine, the mean (+/- SE) lowest systolic and diastolic blood pressure increased significantly from 68.7 +/- 3.1 and 42.8 +/- 2.0 mm Hg to 84.7 +/- 3.9 and 52.7 +/- 2.7 mm Hg respectively during the study (p < 0.01). Midodrine treatment also significantly increased postdialysis systolic and diastolic blood pressures from baseline values of 90.8 +/- 3.8 and 58.3 +/- 3.0 mm Hg to 113.3 +/- 7.1 and 70.6 +/- 3.1 mm Hg (p < 0.01 and p < 0.01 respectively). In addition, oral administration of midodrine also significantly decreased the total volume of intravenous fluid administered during symptomatic hypotension. The clinical signs and symptoms during dialysis were improved in all patients. There were no differences in hemoglobin, serum albumin, urea, creatinine, fasting blood sugar or volume removed per dialysis between both periods of the study. In conclusion, our study has demonstrated that midodrine is a safe and effective treatment for the prevention of recurrent intradialytic hypotension if routinely premedicated before each dialysis session.
Administration, Oral↗