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Biomedical subjects

J Feng

Publications and source records attributed to J Feng.

At least 271 records · Page 15Linked to original sources
PubMed1994

The Caulobacter crescentus FlbD protein acts at ftr sequence elements both to activate and to repress transcription of cell cycle-regulated flagellar genes.

The flagellar genes (fla genes) in Caulobacter crescentus are organized into a regulatory hierarchy of four levels, I-IV, in which transcription of the class III and class IV genes late in the cell cycle from sigma 54-dependent promoters depends on expression of the class II genes above them. Timing of fla gene expression has been attributed to sequential activation and repression by specific transcription factors. Here we report that purified FlbD activates transcription in vitro from the sigma 54-dependent class III flbG promoter and repress transcription from the class II fliF promoter by binding to ftr (flagellar transcription regulator) sequence elements required for their transcriptional regulation in vivo. The FlbD protein makes symmetrical base-specific contacts at three highly conserved guanine nucleotides in each half site of ftr1 and ftr1* at flbG and the single ftr4 site at fliF. The dual function of FlbD in activation of class III genes and repression of the class II fliF promoter is consistent with a central role of FlbD as a switch protein mediating the transition from level II to level III fla gene expression.

Bacterial Proteins↗
PubMed1994

Analysis of amino acid residues in the H5-H6 transmembrane and extracellular domains of Na,K-ATPase alpha subunit identifies threonine 797 as a determinant of ouabain sensitivity.

Several amino acid residues of the alpha subunit of the Na,K-ATPase have been identified which alter ouabain sensitivity. These residues are located in the N-terminal half of the alpha 1 subunit suggesting that this portion of the molecule may represent the binding site for cardiac glycosides. However, not all extracellular and transmembrane regions have been investigated, including the H5-H6 membrane-spanning region. To determine if this region of the alpha subunit contributes to ouabain sensitivity, amino acids which have the potential to form hydrogen bonds were substituted with alanine, a non-hydrogen-bonding amino acid. cDNAs encoding enzyme containing these individual amino acid replacements were expressed in ouabain-sensitive HeLa cells, and the ability of the altered enzymes to confer ouabain resistance was examined. Nineteen amino acid substitutions were investigated. T797A (Thr 797 to Ala) was the only substitution which conferred ouabain resistance to sensitive HeLa cells. Three additional substitutions at this position (T797V, T797S, and T797D) were generated in order to examine the effects of the replacements of Thr 797 on ouabain inhibition of Na,K-ATPase activity. The T797V substitution conferred ouabain resistance, but T797S and T797D substitutions did not. The ouabain-resistant cell lines expressing the T797A and T797V substitutions exhibited Na,K-ATPase activity that was 60 and 70 times more resistant to ouabain than the endogenous HeLa or sheep enzymes. The absence of a hydroxyl group at amino acid 797 may be responsible for the reduced sensitivity of the enzyme with substitutions at this position.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

New killing system controlled by two genes located immediately upstream of the mukB gene in Escherichia coli.

The nucleotide sequence was determined of the region upstream of the mukB gene of Escherichia coli. Two new genes were found, designated kicA and kicB (killing of cell); the gene order is kicB-kicA-mukB. Promoter activities were detected in the regions immediately upstream of kicB and kicA, but not in front of mukB. Gene disruption experiments revealed that the kicA disruptant was nonviable, but the kicB-disrupted mutant and the mutant lacking both the kicB and kicA genes were able to grow. When kicA disruptant cells bearing a temperature-sensitive replication plasmid carrying the kicA+ gene were grown at 30 degrees C and then transferred to 42 degrees C, the mutant cells gradually lost colony-forming ability, even in the presence of a mukB+ plasmid. Rates of protein synthesis, but not of RNA or DNA synthesis, fell dramatically during incubation at 42 degrees C. These results suggested that the kicB gene encodes a killing factor and the kicA gene codes for a protein that suppresses the killing function of the kicB gene product. It was also demonstrated that KicA and KicB can function as a post-segregational killing system, when the genes are transferred from the E. coli chromosome onto a plasmid.

Amino Acid Sequence↗
PubMed1994

Biochemical characterization of epidermal growth-factor receptor in Syrian hamster buccal pouch.

Binding of [125I]-epidermal growth factor (EGF) to the membrane of the adult hamster buccal pouch was specific and protein concentration dependent, and was significantly higher than that of rat buccal tissue (hamster versus rat; 12.1 +/- 0.9 versus 9.77 +/- 0.22 fmol/mg protein, mean +/- SD, n = 5, p < 0.01). The higher binding was due to a higher receptor number (Bmax). Western blot analysis using anti-EGF receptor antibody revealed four immunoreactive protein bands of 170, 150, 116 and 55 kDa in hamster buccal membrane preparations. In a separate experiment, the EGF concentration in the phosphate-buffered saline extract of the hamster submandibular gland was determined by radioimmunoassay; the level was significantly much lower (p < 0.01) than that of rat or mouse (hamster, rat, mouse; 24.2 +/- 2.6, 123 +/- 17, 5950 +/- 171 ng/g wet tissue; mean +/- SD, n = 3). The findings suggest that hamster buccal pouch is particularly well suited to the study of the biological functions of oral EGF receptor.

Animals↗
PubMed1994

Class III antiarrhythmic drug action in experimental atrial fibrillation. Differences in reverse use dependence and effectiveness between d-sotalol and the new antiarrhythmic drug ambasilide.

BACKGROUND: Drug therapy to maintain sinus rhythm in patients with atrial fibrillation (AF) is limited by adverse effects and inadequate efficacy. There has been an increased interest in the use of class III drugs to treat AF, and several new agents have been developed, but there is little information available about mechanisms of class III drug action in AF. The present study was designed to compare the effects of two class III agents, d-sotalol and ambasilide, in dog models of experimental AF. METHODS AND RESULTS: A previously developed dog model of sustained vagotonic AF was used to assess the ability of equal loading doses of d-sotalol and ambasilide (2 mg/kg, followed by maintenance infusions), to terminate AF and prevent its induction. At this dose, ambasilide terminated AF in 12 of 12 dogs and prevented AF induction in 10 of 12 dogs; d-sotalol terminated AF in 1 of 8 dogs (P = .001 versus ambasilide) and prevented AF induction in none of 8 dogs (P = .002). An additional dose of d-sotalol (cumulative load, 8 mg/kg) terminated AF in 7 of 8 dogs and prevented induction in 5 of 8 dogs. In an additional 6 dogs with sterile pericarditis and inducible AF, ambasilide prevented AF induction in all 6. An equal dose of d-sotalol (2 mg/kg) failed to suppress AF induction in any dog, but 8 mg/kg of d-sotalol suppressed AF induction in all. Atrial effective refractory period (AERP) was increased by both drugs. However, the effects of d-sotalol on AERP showed strong reverse use dependence, whereas those of ambasilide did not. Neither ambasilide nor d-sotalol significantly altered conduction velocity, and both increased ventricular refractoriness, with d-sotalol once again showing more reverse use dependence. Effective doses of both agents increased AERP and the wavelength for atrial reentry at rapid rates, slowing atrial activation and terminating the arrhythmia. CONCLUSIONS: The class III drugs d-sotalol and ambasilide terminate AF by increasing AERP and the wavelength for reentry. Ambasilide, which has been reported to block both the rapid and slow components of the delayed rectifier (IKr and IKs), shows less reverse use dependence of effects on refractoriness than the pure IKr blocker d-sotalol, possibly explaining the greater effectiveness of ambasilide at an equal dose level. These results indicate that class III drugs can exhibit different profiles of rate-dependent action on AERP and suggest that it may be possible to develop agents that have more desirable rate-dependent profiles than pure blockers of Ikr.

Aminobenzoates↗
PubMed1994

Early treatment with artemether and praziquantel in rabbits repeatedly infected with Schistosoma japonicum cercariae.

When rabbits infected with 48-52 Schistosoma japonicum cercariae once every wk for 6 times or every other day for 5 times were treated ig with artemether (Art) 10 or 15 mg.kg-1 on d7 after the first infection, followed by the repeated administration of the same dose once every 1-2 wk, a promising effect was seen in the groups treated with Art at higher dose. In another group of rabbits with the same drug administration regimens of praziquantel (Pra) were also used in early treatment, but the initial dose was given on d21 after the first infection. The results showed that Pra given at 40 mg.kg-1 in each administration was more promising than the lower dose of 30 mg.kg-1 especially in group treated at 2 wk intervals. Further study indicated that the presence of adult schistosomes in rabbits increased the effect of Pra not only on 21-day-old schistosomule, but also on 14-day-old schistosomules. The results suggest that Art and Pra could be used in field trial for controlling acute schistosomiasis and decreasing the intensity of schistosomal infection.

Animals↗
PubMed1994

[Effect of artemether on glycogen, protein, alkaline phosphatase and acid phosphatase of Schistosoma japonicum].

When mice infected with Schistosoma japonicum for 32-35 d were treated ig with artemether (Art) at a single dose of 300 mg.kg-1 for 24 h, the glycogen content of female and male schistosomes decreased significantly with reduction rates of about 50%. 72 h after medication, the glycogen reduction rates were 64.1-77.9%. Meantime, the protein content of female and male worms was also decreased, the reduction rates being 68.1% and 49.3%, respectively. In infected mice treated ig with Art at the same dosage for 24 h, the inhibition rates of alkaline phosphatase (AKP) activity in female and male worms were 30% and 25%, respectively. 72 h later, the AKP activity of female worm was further inhibited to 62.3% as compared with the control. Besides, the inhibitory effect of Art on acid phosphatase (ACP) activity of female worm was also more apparent than that of male worm. 72 h after medication, the respective inhibition rates of ACP activity in female and male worms were 75.7% and 47.6%. The results indicated that Art might exert its effect on both carbohydrate and protein metabolism of schistosomes.

Acid Phosphatase↗
PubMed1994

Sensory components controlling bacterial nitrogen assimilation.

In enteric bacteria, the transcription of the Ntr regulon is regulated by a signal transduction system that measures and transmits information on the nitrogen status of the cell. Four of the components of this signal transduction apparatus have been previously identified, and the roles of these are known, to a first approximation, from studies with purified components. The sensor is a uridylyltransferase/uridylyl-removing enzyme (UTase/UR) that controls the uridylylation state of the PII protein. PII indirectly regulates the transcription of the Ntr regulon by acting through the kinase/phosphatase protein NRII. In the absence of unmodified PII, NRII autophosphorylates on a histidine residue, and these phosphoryl groups are transferred to the transcription factor NRI, resulting in the conversion of NRI to the form able to activate transcription. In the presence of PII and NRII, NRI approximately P is rapidly dephosphorylated, preventing the activation of Ntr transcription. This PII-dependent dephosphorylation of NRI approximately P is referred to as the regulated phosphatase activity. In this report, we describe improved methods for the purification of the UTase/UR and PII, and the crystallization of PII. We also present improved methods for the assay of the activities of the UTase/UR protein and PII. The results of our assays indicate that purified PII is effective in eliciting the regulated phosphatase activity, but does not affect the autophosphorylation of NRII or affect the transfer of phosphoryl groups from NRII approximately P to NRI. In addition, we demonstrate that the elicitation of the regulated phosphatase activity by PII is strongly dependent on the ratio of NRI approximately P to NRI, and that the isolated N-terminal domain of NRI, once phosphorylated, is dephosphorylated by the regulated phosphatase activity.

Amino Acid Sequence↗
PubMed1994

Effect of cholinesterase inhibition in vitro by huperzine analogs.

Huperzine (Hup) A and B were first discovered and studied by Chinese. Fourteen analogs of Hup were tested for their anticholinesterase (anti-ChE) activity by colorimetric method using rat erythrocyte membrane and serum as enzyme sources. Hup-A showed the highest anti-AChE potency. The anti-BuChE activity of (-)dihydro and (-)tetrahydro analogs were potent than those of Hup-A. Their anti-AChE activities were comparable to those of physostigmine and galanthamine. Dihydro analog inhibition was of the mixed competitive type with a Ki value of 0.12 mumol.L-1. Tetrahydro analog inhibition was of the competitive type with a Ki value of 0.56 mumol.L-1. They were different from isoflurophate to bond to AChE in a reversible manner.

Acetylcholinesterase↗
PubMed1993

Identification of a functional thyroid hormone response element in the upstream flanking region of the human Na,K-ATPase beta 1 gene.

The human Na,K-ATPase beta 1 subunit gene promoter activity is stimulated by thyroid hormone (T3) in the human intestinal Caco-2 cells. To identify potential cis-acting transcriptional regulatory elements involved in this process, chimeric plasmids containing varying lengths of the 5' flanking region of the human beta 1 Na,K-ATPase gene linked to the firefly luciferase reporter gene were introduced into Caco-2 cells by transient transfection. Analysis of T3-regulated luciferase activity of cells carrying these plasmids, and subsequent use of site-directed mutagenesis revealed that a region from -459 to -438 (relative to the transcriptional start site) is required for the induction of the beta 1 Na,K-ATPase gene by T3. An oligonucleotide containing this sequence from -465 to -433 confers T3 responsiveness to a heterologous promoter. Gel mobility shift assays showed specific binding of nuclear proteins of Caco-2 cells to this region and immunoreactive T3 receptor was identified in one of these complexes. These data demonstrate that there is a cis-acting thyroid hormone responsive element in the 5' flanking region of the human beta 1 Na,K-ATPase gene and induction of transcription of this gene by T3 involves specific binding of the thyroid hormone receptor to the TRE located at position -459 to -438.

Adenocarcinoma↗
PubMed1993

Cytogenetic clonality in myelodysplastic syndromes studied with fluorescence in situ hybridization: lineage, response to growth factor therapy, and clone expansion.

Clonality in myelodysplastic syndromes (MDS) has been studied with various techniques including glucose-6-phosphate dehydrogenase (G6PD) isoenzyme and cytogenetic analyses, and with molecular techniques such as gene deletion studies and the analysis of restriction fragment-length polymorphisms (RFLP) of X-linked genes. In this study, we investigated the use of fluorescence in situ hybridization (FISH) with a chromosome-specific probe to examine cytogenetic clonality in peripheral blood (PB) cells from three patients with MDS. In each case, trisomy 8 was shown by conventional cytogenetic analysis at the time of the initial diagnosis. By using FISH with a probe for the centromere of chromosome 8, we identified the trisomy in individual PB cells from Wright-stained smears. With this technique, we could determine the cell lineage involved by the trisomy, and through serial analyses we could assess the response of the clonal and nonclonal cells to growth-factor therapy, and the expansion of the trisomic clone over time. In each of the three cases, various proportions of granulocytes, monocytes, eosinophils, and basophils showed trisomy 8 by FISH analysis. In none of the cases did we detect trisomy 8 in lymphocytes. By analysis of PB cells before and during therapy with recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), we found that GM-CSF stimulated both trisomic and disomic cells. During a 1-year period of sequential study, we detected an abrupt increase in the percentage of trisomic cells in one patient, a stable percentage in another, and a slowly increasing percentage in the third. The abrupt increase in the first patient preceded a transformation to a more acute phase by 2 months. We conclude that FISH analysis of PB cells of patients with MDS offers an additional approach to the study of clonality in this disorder. In some cases this analysis may provide a useful and simple means of assessing response to therapy and progression of disease.

Aged↗
PubMed1993

The fate of human peripheral blood lymphocytes after transplantation into SCID mice.

Human peripheral blood lymphocytes (hu-PBL) can be adoptively transferred by intraperitoneal injection into mice with severe combined immunodeficiency (SCID). The transplanted lymphocytes can produce immunoglobulin (Ig), respond to antigens, and survive for months in this chimeric model (hu-PBL SCID). However, whether the lymphocytes actually repopulate and reconstitute lymphoid structures and organs has been subject of some debate. To address this question and to characterize the hu-PBL SCID model better, we employed a novel technique for the identification of human cells in xenogeneic mice. We used fluorescence in situ hybridization (FISH) with a biotinylated DNA probe to all human centromeres. We demonstrated that FISH could be used to detect human cells when they accounted for less than 1% of human/mouse cell mixtures; it could also be employed for the identification and localization of individual human cells in tissue sections. By using FISH, we studied 31 SCID mice injected with 1.5 x 10(7)-4 x 10(7) hu-PBL via intravenous (i.v.) or intraperitoneal (i.p.) routes. In the 6 i.v.-injected mice, we found that the human cells were removed from the circulation into the lung within 1 h. In 22 of 25 i.p.-injected animals, 90-3716 micrograms/ml of human IgG was found in the sera at 3 to 13 weeks after transplantation (a.t.). Human cells colonized the peritoneal cavity and persisted for up to 13 weeks a.t. and, in the 12 mice studied, accounted for 4% to 57% of the cells in the peritoneal fluid. However, only rare, isolated human cells were found in the spleen, blood, bone marrow, lung or Peyer's patches. In 7 of 19 mice that received hu-PBL i.p. from Epstein-Barr virus-seropositive donors, we found masses of human cells usually beneath the peritoneal lining but sometimes infiltrating normal tissue. We conclude that FISH offers a simple means for accurate identification of human cells in the xenogeneic mouse. Although there is colonization of the peritoneal cavity in most mice, and development of lymphoid masses in some, there is no reconstitution of lymphoid structures and only minimal engraftment of lymphoid organs by human cells in conventionally-prepared hu-PBL SCID constructs.

Animals↗