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Biomedical subjects

L Chen

Publications and source records attributed to L Chen.

At least 1,261 records · Page 70Linked to original sources
PubMed1993

Site-specific mutagenesis of the metal binding sites of porcine fructose-1,6-bisphosphatase.

Mutations in the metal binding sites of porcine fructose-1,6-bisphosphatase were carried out by site-specific mutagenesis based on the crystal structure of the enzyme. The mutant and wild-type enzymes have been characterized by circular dichroism spectrometry and initial-rate kinetics. One of the mutant forms of fructose-1,6-bisphosphatase (Glu280Gln) is associated with a single metal site, whereas two other mutants (Glu97Gln and Asp118Asn) ligate two metal ions. The mutant enzymes exhibit very large decreases in kcat relative to the wild-type enzyme; however, other kinetic parameters, such as Km values, are not greatly altered. Metal binding cooperativity and binding affinity is decreased in the mutants compared to wild-type fructose-1,6-bisphosphatase. Mutations in the metal binding sites greatly enhance the enzyme's affinity for AMP, a potent regulator of fructose-1,6-bisphosphatase activity. The results of these investigations are fully consistent with predictions made on the role of specific amino acid residues at the metal binding sites in porcine fructose-1,6-bisphosphatase from X-ray diffraction studies [Zhang, Y., Liang, J.-Y., Huang, S., Ke, H., and Lipscomb, W.N. (1993) Biochemistry, 32, 1844-1857].

Animals↗
PubMed1993

Complete genomic sequence and phylogenetic analysis of the lactate dehydrogenase-elevating virus (LDV).

The apparently complete sequence of the RNA genome of the neurovirulent isolate of lactate dehydrogenase-elevating virus (LDV-C) has been determined. The LDV-C genome is at least 14,222 nucleotides in length and contains eight open reading frames (ORFs). ORF 1a, which encodes a protein of 242.8 kDa and is located at the 5' end of the genome, contains at least two putative papain-like cysteine protease domains, and one putative chymotrypsin-like serine protease domain. This ORF terminates with a UAG stop codon that can be bypassed if a -1 frameshift occurs. The frameshift region consists of a heptanucleotide "slippery" sequence, 5'-UUUAAAC-3', followed by a putative pseudoknot. ORF 1b encodes a protein of 155.4 kDa containing, in its N-terminal portion, an RNA-dependent RNA polymerase and an RNA helicase domain separated by a Zn finger domain. Another domain of unknown function that is also conserved in coronaviruses and toroviruses is located at the C-terminus of the ORF 1b product. Three cleavage sites in the ORF 1a polyprotein and three in the ORF 1b polyprotein were predicted for the chymotrypsin-like protease and tentatively delimit the mature nonstructural proteins of LDV. Six small, overlapping 3' ORFs (ORFs 2 through 7) encode proteins with calculated sizes of 25.8, 21.6, 19.8, 23.9, 18.9, and 12.3 kDa. ORF 7 encodes the virion nucleocapsid protein Vp-1, while ORF 6 encodes the nonglycosylated envelope protein Vp2. ORFs 5, 4, 3, and 2 each encode glycoproteins which may be virion envelope proteins. LDV is closely related to equine arteritis virus, Lelystad virus (LV), and simian hemorrhagic fever virus. These four viruses belong to a new group of positive-strand RNA viruses and are related to coronaviruses and toroviruses.

Amino Acid Sequence↗
PubMed1993

Metastatic atrial myxoma presenting as intracranial aneurysms with hemorrhage: case report.

A 68-year-old woman, developed subsequent recurrent hematomas of the left occipital lobe about 1 year after open-heart surgery for the left atrial myxoma. Radiological studies revealed multiple intracranial aneurysms with hemorrhage. Microscopic examination showed the presence of myxoma invasion of the vascular wall with aneurysmal formation and organized hematoma.

Aged↗
PubMed1993

Costimulation of T cells for tumor immunity.

Tumor specific antigens can be demonstrated on many neoplasms by immunization and challenge experiments; however, these antigens do not normally elicit a sufficiently strong immune response to prevent tumor growth in immunocompetent hosts. Recent studies have demonstrated that efficient activation of T cells requires costimulation of the CD28 receptor via the B7 molecule on antigen-presenting cells. Inadequate costimulation of tumor-reactive T cells may contribute to the fact that antigenic tumors are not normally rejected by the immune system, and weak anti-tumor immune responses may be amplified by upregulation of CD28 triggering.

Animals↗
PubMed1993

Histological changes in rat liver tumours treated with high-intensity focused ultrasound.

This paper reports the histological changes found in rat liver tumours treated with high-intensity focused ultrasound. HSN fibrosarcoma, implanted subcapsularly in the livers of CBH rats, were treated using an array of ultrasound exposures. At predetermined times following treatment, the rats were sacrificed and tissue specimens were examined histologically. Evident tissue damage was confined to regions that had been given high ultrasound exposures. Within these regions ("lesions") there was no evidence of intact cells whereas in the sharply demarcated surrounding tissue there was no evidence of cell damage. Where individual ultrasound lesions had been placed in sufficiently close proximity, there was correspondingly continuous and complete cell destruction. There is suggestive evidence that tissue damage may arise through two different mechanisms: direct, primarily thermal, damage and indirect damage resulting from compromised blood supply. Under the same exposure conditions, normal liver cells appear to lose their morphological structure more readily than do tumour cells.

Animals↗
PubMed1993

Recovery of CAP threshold and amplitude in chickens following kanamycin ototoxicity.

Chickens were given a dose of kanamycin (400 mg/kg/d x 10 d) which destroyed hair cells over the basal 37-58% of the basilar papilla. Afterwards, the threshold and amplitude of the compound action potential were measured at recovery times ranging from 2 days to 10-20 weeks post-kanamycin treatment. At 2 days post-treatment, the thresholds at 1000, 2000 and 4000 Hz were elevated 40-60 dB while the thresholds at 250 and 500 Hz were elevated only 25 dB. By 10-20 weeks post-treatment, the threshold at 250 and 500 Hz had completely recovered whereas a residual threshold shift of 5 dB to 25 dB was present between 1000 to 4000 Hz. The maximum amplitude of the compound action potential was also reduced by more than 60% at all frequencies at 2 days post-treatment; however by 10-20 weeks post-treatment, the amplitude of the compound action potential had completely recovered at 500, 1000 and 2000 Hz. By contrast, the amplitude of the compound action potential at 4000 Hz was still reduced by more than 50% of its normal value 10-20 weeks post-treatment. The results of the present study indicate that the time course of recovery of the compound action potential is extremely slow and may lag behind the regeneration of hair cells by many weeks. The permanent deficits observed at the high frequencies could conceivably be due to functional deficits in regenerated hair cells, their afferent synapses or the loss of cochlear ganglion cells.

Acoustic Stimulation↗
PubMed1993

Determination of peptide 520 in human plasma using post-column photolysis with electrochemical detection in liquid chromatography.

A simple LC method for the determination of peptide 520 in human plasma was developed. Based on micellar chromatography, sodium octyl sulphate (SOS) was added into the mobile phase in order to separate the peptide from human plasma components. The procedure was fast and sensitive for the determination of the peptide in untreated human plasma. The electrochemical (EC) detection limit for peptide 520 in human plasma was 0.5 microgram ml-1. Linearity of the calibration plot for peptide 520 in human plasma was 0.999. This approach represents a direct injection technique for the potential detection and analysis of numerous peptides in biofluids, besides just plasma, with absolute quantification.

Chromatography, Liquid↗
PubMed1993

Organic-phase biosensors for monitoring phenol and hydrogen peroxide in pharmaceutical antibacterial products.

Organic-phase biosensors open new opportunities for assays of challenging pharmaceutical products. Such opportunities are illustrated for the rapid determination of phenol and peroxide antiseptics in different anti-infective formulations. The tyrosinase and peroxidase enzyme electrodes offer reliable quantification of these antibacterial agents following sample dissolution in the organic solvent. The dynamic properties of these enzyme electrodes are exploited for rapid and reproducible flow-injection assays of the pharmaceutical products (relative standard deviation = 1.6-1.9%). Such developments should facilitate rapid quality control testing in the pharmaceutical industry and should be applicable to other therapeutic agents and products. Applicability to cosmetic products containing hydrogen peroxide is also demonstrated.

Anti-Infective Agents, Local↗
PubMed1993

Effect of blood perfusion on the ablation of liver parenchyma with high-intensity focused ultrasound.

This paper discusses the effect of blood perfusion on the ablation of rat liver tissue with high-intensity focused ultrasound (HIFU). For this study a practical method has been developed, in which the liver blood flow can be reduced by ligation of the hepatic artery and portal vein. During the treatment the rat liver was mobilized out of the abdomen and the blood flow was measured using both the radioactive microsphere method and a laser Doppler blood-flow monitor. The results show that the hepatic blood flow was about 23 ml/100 g min-1 via the hepatic artery and about 227 ml/100 g min-1 via the portal vein. The total liver blood flow was reduced by 98% when both the hepatic artery and portal vein were ligated. Comparative lesions were made on the same liver lobes of rats with both normal and reduced blood flow using a focused ultrasound beam of 1.7 MHz, 67-425 W cm-2 spatially averaged focal intensity ISAL and 2-20 s exposure duration. A marked difference has been found between the lesion dimensions obtained with normal blood flow and that with reduced blood flow. For exposures at 169 W cm-2 the lesion diameter with normal blood flow was reduced by 14% for 3 s exposure duration compared to that obtained with both hepatic artery and portal vein ligated, while the reduction was more than 20% for longer durations.

Animals↗
PubMed1993

Preferences for decision-making autonomy.

Nurses' lack of autonomy has been identified as a leading cause of job dissatisfaction, but attempts to increase satisfaction by increasing autonomy have not always been successful. This survey of 356 randomly selected staff nurses and 130 head nurses from 16 hospitals extends previous work by identifying the preferred level of involvement in 21 patient care and 21 unit operation decisions. Staff nurses agreed on 60 percent of the decisions and, in general, preferred independent decision-making for patient care decisions and shared decision-making for unit operation. Head nurses indicated that staff nurses should have a higher level of autonomy than the staff nurses indicated for themselves.

Adult↗
PubMed1993

Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions.

We have cloned two metal-regulated genes (mrgA and mrgC) from Bacillus subtilis by using transposon Tn917-lacZ. Both were isolated as iron-repressible gene fusions, but the metal specificity and sensitivity of gene repression are distinct. Transcription of mrgA-lacZ is induced at the end of logarithmic-phase growth in minimal medium, and this induction is prevented by excess manganese, iron, cobalt, or copper. Limitation for metal ions is sufficient for mrgA-lacZ induction, since resuspension in medium lacking both manganese and iron rapidly induces transcription. Transcription of mrgC-lacZ is also induced by iron deprivation but is not repressed by added manganese or other metal ions. Expression of mrgC-lacZ and a 2,3-dihydroxybenzoic acid-based siderophore is repressed in parallel by iron, and in both cases, only iron effects repression. We have cloned and sequenced the promoter and regulatory regions of both mrgA and mrgC. Both genes are preceded by a predicted sigma A-dependent promoter element with overlapping sequences similar to the iron box consensus element for recognition by the Escherichia coli ferric uptake regulator protein (Fur). Mutation of the putative iron box for gene mrgC leads to partial derepression in iron-replete medium.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis.

A genetically defined mutation, designated rfa-2, results in altered lipopolysaccharide (LPS) biosynthesis. rfa-2 mutants produce a core-defective LPS that contains lipid A and a single sugar moiety, 2-keto-3-deoxyoctulosonic acid, in the LPS core region. Such LPS core-defective or deep-rough (R) mutant structures were previously designated chemotype Re. Phenotypically, rfa-2 mutants exhibit increased permeability to a number of hydrophilic and hydrophobic agents. By restriction analyses and complementation studies, we clearly defined the rfa-2 gene on a 1,056-bp AluI-DraI fragment. The rfa-2 gene and the flanking rfa locus regions were completely sequenced. Additionally, the location of the rfa-2 gene on the physical map of the Escherichia coli chromosome was determined. The rfa-2 gene encodes a 36,000-dalton polypeptide in an in vivo expression system. N-terminal analysis of the purified rfa-2 gene product confirmed the first 24 amino acid residues as deduced from the nucleotide sequence of the rfa-2 gene coding region. By interspecies complementation, a Salmonella typhimurium rfaC mutant (LPS chemotype Re) is transformed with the E. coli rfa-2+ gene, and the transformant is characterized by wild-type sensitivity to novobiocin (i.e., uninhibited growth at 600 micrograms of novobiocin per ml) and restoration of the ability to synthesize wild-type LPS structures. On the basis of the identity and significant similarity of the rfa-2 gene sequence and its product to the recently defined (D. M. Sirisena, K. A. Brozek, P. R. MacLachlan, K. E. Sanderson, and C. R. H. Raetz, J. Biol. Chem. 267:18874-18884, 1992), the S. typhimurium rfaC gene sequence and its product (heptosyltransferase 1), the E. coli K-12 rfa-2 locus will be designated rfaC.

Amino Acid Sequence↗
PubMed1993

Phenotypic effects of targeted mutations in the small subunit rRNA gene of Tetrahymena thermophila.

Tetrahymena thermophila is an ideal organism with which to study functional aspects of the rRNAs in vivo since the somatic rRNA genes of T. thermophila can be totally replaced by cloned copies introduced via microinjection. In this study, we made small insertions into seven sites within the small subunit rRNA gene and observed their phenotypic effects on transformed cells. Two mutated genes coding for rRNA (rDNAs), both of which bear insertions in highly conserved sequences, failed to transform and are therefore believed to produce nonfunctional rRNAs. Three other altered rDNAs produce functional rRNAs that can substitute for most or all of the cellular rRNA. Two of these bear insertions in highly variable regions, and, surprisingly, the other has an insertion in a region that is well conserved for both sequence and secondary structure among eucaryotes. In addition, two other insertions appear to destabilize rRNAs that contain them. Our findings make predictions concerning the positions of some of these sites within the tertiary structure of the small ribosomal subunit and thus serve as an in vivo test of the existing tertiary structure models for the small subunit rRNA. Our results are in good agreement with expectations based on sequence comparison and in vitro work.

Animals↗
PubMed1993

Structure, mapping, and expression of erp, a growth factor-inducible gene encoding a nontransmembrane protein tyrosine phosphatase, and effect of ERP on cell growth.

We have characterized a growth factor-inducible gene, erp, and demonstrated that it encodes a 367-amino-acid nontransmembrane tyrosine phosphatase protein with significant similarity to the vaccinia virus H1 protein. Immunoprecipitation analyses show that the erp protein, ERP, is rapidly induced following serum stimulation of quiescent fibroblasts. ERP has been expressed as a fusion protein with glutathione S-transferase and shown to have tyrosine as well as serine protein phosphatase activity. The enzymatic activity of ERP depends on the presence of reducing agents such as dithiothreitol, and its tyrosine phosphatase activity is inhibited by sodium vanadate, a potent inhibitor of protein tyrosine phosphatases. The number of stable NIH 3T3 clones obtained after transfection with a vector expressing the complete ERP protein is reduced more than 90% compared with that after transfection with a vector expressing a mutated inactive ERP protein. The remaining ERP-expressing clones present a significant increase in the proportion of bi- and multinucleated cells and a decrease in proliferation rate. Studies on the genomic structure reveal that the erp transcription unit is 2.8 kbp long and split into four exons. The erp gene maps to the 17A2-17C region of the murine genome. Our results demonstrate that the protein product of the immediate-early gene erp has a negative effect on cell proliferation.

3T3 Cells↗
PubMed1993

Altering creatine kinase isoenzymes in transgenic mouse muscle by overexpression of the B subunit.

To change the levels of expression and isoenzyme distribution of creatine kinase (CK) in muscle, transgenic technology was used to express the B subunit of CK in mouse muscle. Normally, mammalian skeletal muscle contains the MM dimer of CK. The BB dimer and MB heterodimer of CK can be found in brain and heart, respectively. Heterologous genes consisting of skeletal and cardiac muscle-specific actin promoters fused to the genomic coding region of the B form of CK were used to create transgenic mice. Lines were established from the three highest expressing founders. Analysis of skeletal muscle extracts revealed that all three lines had an increase in total CK activity measured under maximal velocity conditions. The highest expressing line, 7001, had a CK activity 150% that of control muscle. Nuclear magnetic resonance saturation transfer was used to measure the in vivo rate of the CK reaction. In 7001 hindlimb muscles, the CK catalyzed reaction was 200% that of control muscle. The elevation in CK activity in transgenic muscle was accompanied by significant changes in the composition of the cytosolic isoenzyme ratio of CK. In control, 100% of CK was MM, whereas 7001 had 60 +/- 18% MM, 32 +/- 10% MB, and 8 +/- 2% BB. There were no changes in ATP, phosphocreatine, Pi, or creatine levels in transgenic muscle compared with control. Immunofluorescence of myofibrils isolated from control and transgenic muscle revealed specific association of CK to the M line. Small amounts of MB CK were detected on myofibrils from transgenic mice. Transgenic mice expressing the B subunit of CK in muscle represent a first step toward altering CK isoforms so as to elucidate the specific roles of these isoforms in energy metabolism.

Animals↗