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Biomedical subjects

J Pan

Publications and source records attributed to J Pan.

At least 325 records · Page 18Linked to original sources
PubMed1977

Comparison of the nucleotide sequence of the messenger RNA for the major structural protein of SV40 with the DNA sequence encoding the amino acids of the protein.

The 16S late mRNA from SV40 directs the synthesis of the major viral structural protein, VP1. We have compared the oligonucleotides in the 16S mRNA with those that would be present in a transcript of the portion of SV40 DNA coding for VP1. The results indicate that a segment of about 200 nucleotides of RNA transcribed from a distant part of SV40 DNA have become linked to the transcript of VP1 codons by a bond resistant to phenol extraction and denaturation in formamide.

Base Sequence↗
PubMed1976

SV40 DNA sequences as an example of the structure of genes functioning in animal cell nuclei.

Recent studies of the structure of messenger RNA have demonstrated the existence of untranslated sequences of the 3' and 5' end of the messages. In addition analysis of transcription in vitro has indicated that the nucleotide sequence U6 purine may be part of a transcription termination signal in prokaryotes. Recently it has been possible to determine the sequence of extensive portions of the DNA of SV40 virus. This article reviews the analogies between certain of these sequences and sequences available from prokaryotic messengers and DNAs. Unusual structures, including blocks of AT-rich and GC-rich segment sections and symmetric regions in the DNA near the origin of DNA replication, have been demonstrated and the distribution of stretches of 6 or more deoxyadenylic acids in the DNA of SV40 is consistent with some rho for these sequences in animal cells, either as terminators of transcription or as sites where degradation of transcripts is initiated or sites related to the selective rejection or degradation of transcipts.

Animals↗
PubMed1974

Nucleotide sequences of RNA transcribed in infected cells and by Escherichia coli RNA polymerase from a segment of simian virus 40 DNA.

The nucleotide sequence of 180 residues of an RNA transcript of DNA of simian virus 40 has been deduced. This sequence adjoins a preferred initiation site for E. coli RNA polymerase. Comparison of this sequence with that of complementary RNA of simian virus 40 from infected cells has shown that this site also adjoins the apparent 3' terminus of some of the cytoplasmic complementary RNA of simian virus 40.

Adenoviridae↗
PubMed1974

The nucleotide sequence preceding an RNA polymerase initiation site on SV40 DNA. Part 1. The sequence of the late strand transcript.

The nucleotide sequence of the transcript of the "late" strand of the region of SV40 DNA preceding the preferred initiation site for Escherichia coli RNA polymerase has been determined to be U-G-U-A-A-C-C-A-U-U-A-U-A-A-G-C-U-G-C-A-A-U-A-A-A-C-A-A-G-U-U-A-A-C-A-A-C-A-A-C-A-A-U-U-G-Cp. Hemophilus influenza restriction endonuclease cleaves this region 30 nucleotides (base pairs) before the site of initiation of RNA synthesis by RNA polymerase.

Base Sequence↗
PubMed1974

The nucleotide sequence preceding an RNA polymerase initiation site on SV40 DNA. Part 2. The sequence of the early strand transcript.

The nucleotide sequence of the RNA transcript from the "early" (E) strand of SV40 DNA immediately preceding the preferred E. coli RNA polymerase start site is G-(A-A-A-C, -A-U-)-A-A-A-A-U-G-A-A-U-G-C-A-A-U-U-G-U-U-G-U-U-G-U-U-A-A-C-U-U-G-U-U-U-A-U-U-G-C-A-G-C-U-U-A-U-A-A-U-G-G-U-U-A-C-Ap. The last nucleotide of the sequence is the first nucleotide transcribed by E. coli RNA polymerase from the "E" strand. The DNA template contains a palindrome of 17 residues that includes the Hemophilus influenza restriction endonuclease cleavage site G-T-T-A-A-Cp. The DNA which gives this transcript lies very close to one end of SV40 DNA segment in the Adeno-SV40 hybrid virus Ad2+ND3 and appears to contain sufficient untranscribed information to specify the E. coli RNA polymerase start.

Base Sequence↗
PubMed1974

The mapping and ordering of fragments of SV40 DNA produced by restriction endonucleases.

SV40 DNA is cleaved by the Eco RII and Hae restriction endonucleases to give rise to two different sets of 16 fragments each. These fragments have been ordered by analysis of the products of redigestion of one set of fragments with another restriction enzyme. The cleavage sites have been precisely mapped based on length measurements of the products of each cleavage. This provides a convenient group of small DNA fragments suitable for sequence analysis investigation of the transcripts present in infected cells, or construction of deletion substitution variants of the virus.

DNA Restriction Enzymes↗
PubMedDate not supplied

High-throughput determination of identity, purity, and quantity of combinatorial library members using LC/MS/UV/ELSD.

We have used a high-throughput LC/MS/UV/ELSD method to rapidly determine the absolute quantity and purity of 42 organic compounds from seven lead discovery libraries. A general calibration curve generated from a different set of 42 compounds with seven different scaffolds was used in this analysis. We have also studied 33 organic compounds with different molecular weight (MW) by LC/MS/UV/ELSD to investigate the effect of MW on ELSD response and the accuracy for purity and quantity measurement using UV(214) and ELSD. A general ELSD calibration curve from these compounds was also generated to quantify 42 library compounds. Purity measurement by ELSD underestimates the amounts of impurities due to a reduced ELSD response from smaller molecular weight impurities often produced in library synthesis. Absolute quantity determination by ELSD is more accurate (RSD 28%) than that by UV(214) (48%) using a calibration curve generated from the same set of compounds with diverse MWs. Error assessment for the measurement of absolute quantity of a class of commercial compounds and a class of representing reference compounds from seven diverse lead discovery libraries shows that structurally related compounds should be used to generate calibration curves to sustain smaller deviation.

Calibration↗
PubMedDate not supplied

Evaluation of evaporative light-scattering detector for combinatorial library quantitation by reversed phase HPLC.

A quantitation study using reversed phase HPLC with UV and evaporative light-scattering detector (ELSD) was conducted on 90 library standards selected from 15 small molecule combinatorial libraries (six standards from each library). This study assessed the quantitation errors using a single calibration curve for rapid purity analysis of combinatorial libraries. The average quantitation error of six standards from one library at 200 microM by UV was 13. 4%, 20.6%, and 60.3%, at 214, 220, and 254 nm, respectively. By ELSD, the average quantitation error of these six standards at 200 micro was only 7.7%. Applying this ELSD calibration curve to 84 standards from 14 structurally diverse libraries, an average quantitation error of 16.4% was obtained. The average quantitation error of all 90 standards from 15 libraries using 15 calibration curves was 18.5%.

Calibration↗
PubMedDate not supplied

A point mutation in the A gamma-globin gene promoter in Greek hereditary persistence of fetal haemoglobin.

Hereditary persistance of fetal haemoglobin (HPFH) is a benign condition characterized by the production in adulthood of more than 1% fetal haemoglobin (HbF, alpha 2 gamma 2) in the absence of erythropoietic stress. Several genetic types have been discerned based on the level of HbF produced, the relative contributions of the duplicated fetal (G gamma and A gamma) globin genes, and the presence or absence of deletions involving the beta and delta genes in cis to the mutation. Greek HPFH is a non-deletion variety in which heterozygotes produce 10-20% HbF, predominantly due to overproduction of the A gamma chain. We have cloned a 40-kilobase (kb) region of the beta-globin cluster from a Greek HPFH allele and report here that a point mutation (G----A) occurs 117 base pairs (bp) 5' to the cap site of the A gamma-globin gene, just upstream of the distal CCAAT sequence. The corresponding region of the G gamma-globin gene is normal. We discuss the implications of this finding for the developmental regulation of globin gene expression.

Base Sequence↗
PubMedDate not supplied

Polymorphism of metabolic genes and susceptibility to occupational chronic manganism.

In this study we investigated genetic polymorphisms of five metabolizing genes and their association with occupational chronic manganism. We recruited 49 patients with chronic manganism and 50 unrelated healthy control subjects who were welders and ferromanganese smelters and occupationally exposed to manganese dust and fume in the same workshops from three metallurgical industries. The controls were matched to the cases by sex, age, cigarette and alcohol intake, as well as the manganese exposure duration. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to genotype the cytochrome P450 2D6L gene (CYP2D6L) and the NAD(P)H:quinone oxidoreductase gene (NQO1). Allele-specific PCR was used to detect the cytochrome P450 1A1 gene (CYP1A1), and the glutathione-S-transferase mu and theta genes (GSTM and GSTT). The frequency of polymorphic alleles, a mutation of CYP2D6L, was significantly lower in patients with chronic manganism (16.3%) than in controls (29.0%). Individuals with the homozygote polymorphism (L/L) of CYP2D6 had a 90% decreased risk of chronic manganism compared with the wild-type (Wt/Wt) (odds ratio =0.10, 95% confidence interval = 0.01-0.82). A significant association between the CYP2D6 genotype subgroup and the latency of chronic manganese poisoning was also found. Patients who had homozygous (L/L) or heterozygous (Wt/L) mutant alleles developed manganism an average of 10 years later than those who were homozygous wildtype (Wt/Wt). However, the allele and genotype frequencies of CYP1A1 and NQO1 genes were distributed similarly in cases and controls. In addition, no difference in the frequencies of GSTM1 and GSTT1 null genotypes were observed between cases and controls. The results suggest that CYP2D6L gene polymorphism might influence susceptibility to manganese-induced neurotoxicity. However, because of limited sample size, our results should be validated in large-scale studies.

Adult↗
PubMedDate not supplied

Clinical significance of gastric myoelectrical dysrhythmias.

Normal rhythmic myoelectrical activity of the human stomach is 3 cpm, regulating gastric contractile activity. Dysrhythmia in gastric myoelectrical activity is found to be associated with functional disorders of the stomach. Gastric dysrhythmias are classified into tachygastria (frequency higher than normal), bradygastria (frequency lower than normal) and arrhythmia (no rhythmic activity). Clinical significance of gastric dysrhythmias is discussed in this paper, outlined as follows: (a) What is normal gastric myoelectrical activity and what is dysrhythmia? (b) How to detect gastric dysrhythmias? (c) Gastric dysrhythmias in clinical settings. (d) What may cause gastric dysrhythmias? (e) How to normalize gastric dysrhythmias?

Electrodiagnosis↗
PubMedDate not supplied

Isolation and culture of porcine hepatocytes for artificial liver support.

The primary requirement of cells in a liver support system is the preservation of the in vivo metabolic functions that prevent or decrease the progress of hepatic encephalopathy (HE) by providing interim support to liver failure patients. While rodent hepatocytes offer a model for liver assist device (LAD) research, their limited number per animal prohibits direct scale up to human devices. Healthy human liver cells are seldom available in adequate numbers to support clinical LAD use; consequently, a large animal source of liver cells is needed. The study presented here explored the potential of porcine hepatocytes to proliferate and maintain metabolic function in vitro. Porcine hepatocytes were isolated from approximately 12 kg swine by a modification of Seglen's method. Hepatocytes cultured up to 10 days were shown to metabolize ammonia and maintain both Phase I and II detoxification functions. In addition, the cultures showed proliferative activity both as an increase in total protein content and by thymidine incorporation. Immunocytochemical staining identified cell proliferation through Day 4 to be primarily hepatocytes while Days 6 and 10 showed nonparenchymal cells to be increasing. The detoxification functions measured showed peak activity on Day 4 and gradually declined through Day 10. The ability of porcine hepatocytes to proliferate and maintain a diversity of hepatic functions in culture strongly suggests their potential for use as the biological component of artificial LADs.

Acetaminophen↗
PubMedDate not supplied

Functional recovery of porcine hepatocytes after hypothermic or cryogenic preservation for liver support systems.

The provision of an immediate supply of isolated porcine hepatocytes for artificial liver support requires preservation techniques that will allow maintenance of cell viability and detoxification functions. By means of a simple and cost-effective cryopreservation system, porcine hepatocytes can be available for both local and distant medical treatment facilities. Additionally, cryopreservation provides an adequate period for quality control testing to be completed prior to use of any specific cell lot. We are reporting a dual approach, namely the preservation of porcine hepatocytes, at 4 degrees C and at -196 degrees C in liquid nitrogen (LN2). Using a combination of cryoprotectant agents with Chee's modified Eagle's culture media (CEM), collagenase isolated hepatocytes stored at 4 degrees C for 24 h maintained 80% of the initial diazepam metabolism measured in freshly isolated cells and nearly 100% of initial function was preserved in hepatocytes stored up to 6 mo at -196 degrees C. University of Wisconsin solution (UW) was also tested and while adequate for 4 degrees C storage, it certainly did not match the performance of the CEM formulations for preservation of metabolic function of cells stored in liquid nitrogen. Based on our results of viability and detoxification function the combination of CEM with DMSO, polyethylene glycol and serum provided optimal protection for LN2 frozen cells. Other findings in these studies underlined the importance of the gradual introduction of DMSO in the prefreezing process, the period of osmotic equilibration, and the rapid postthaw withdrawal of this agent to minimize cytotoxic effects at these critical stages. Our freezing methodology provides the foundation for further technological developments in the cryopreservation of the large numbers of cells (billions) that are necessary for extracorporeal liver assist devices.

Animals↗
PubMedDate not supplied

Characterization and evaluation of detoxification functions of a nontumorigenic immortalized porcine hepatocyte cell line (HepLiu).

Primary porcine hepatocytes (PPH) are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices to overcome the shortage of human hepatocytes. However, their finite life span and typically rapid loss of functions limit their utility. An immortalized, nontumorigenic, highly differentiated porcine hepatocyte cell line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control of the SV40 early promoter. From the established 69 clones, 23 clones displaying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for > 2 years, through more than 60 passages and 240 divisions. Albumin protein, present in early passages, was lost at later passages, but albumin transcript still was detectable in later passages. Carbamoyl phosphate synthetase, a gateway enzyme of the urea cycle, was consistently detectable in HepLiu cells. Cytokeratin 18, a characteristic marker of primary hepatocytes, was detected by both immunofluorescent staining and Western blot in HepLiu cells. Furthermore, maintenance of P450 functions in HepLiu cells was evidenced by diazepam and 7-ethoxycoumarin metabolites measured by HPLC. Phase II conjugative function was measured as acetaminophen glucuronidation. P450 dealkylase was demonstrated microscopically by the conversion of a nonfluorescent substrate to a fluorescent product. Both Northern blot analysis and immunofluorescent staining showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined immunodeficient (SCID) mice nor was the TAI (a tumor marker) mRNA expressed, even in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, liver biologic regulation studies, and artificial liver support systems.

Animals↗