Biomedical subjects

R Chen

Publications and source records attributed to R Chen.

At least 289 records · Page 16Linked to original sources

PubMed1998

Induction of microsatellite instability by oxidative DNA damage.

Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer. Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development. To study the source of this instability, we have identified a region of the beta-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance. Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of beta-lactamase can be selected on the basis of antibiotic resistance. We have utilized this site to insert a synthetic microsatellite sequence within the beta-lactamase gene and selected for mutations yielding frameshifts. This assay provides for detection of one frameshift mutation in a background of 10(6) wild-type sequences. Mismatch repair deficiency increased the observed frameshift frequency approximately 300-fold. Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N'-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis. We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences. This beta-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.

Base Sequence↗

PubMed1998

Effect of neutrophil depletion in acute cerebritis.

Inhibition of the host's neutrophil response has been proposed as one means to reduce tissue damage in acute inflammation. If this approach can be applied in acute central nervous system (CNS) infection, the long-term morbidity, which occurs in CNS infection, might be reduced. Previous studies in models of CNS infection yielded conflicting results whether neutrophil depletion might be protective. To determine whether neutrophil depletion reduces tissue necrosis and cerebrovascular injury in experimental bacterial cerebritis, we depleted circulating neutrophils with an IgM monoclonal antibody, RP3, given after the start of the infection. RP3 treatment successfully depleted circulating neutrophils and reduced the extent of neutrophil influx into the cerebritis region. The extent of tissue necrosis, measured histologically, and the regional increase of blood-brain barrier (BBB) permeability were not inhibited by neutrophil depletion, and in animals treated with RP3 alone, the extent of tissue necrosis and BBB permeability tended to be larger than in S. aureus inoculated controls. We conclude that host neutrophils do not add to the tissue and cerebrovascular damage created by the intracerebral inoculation of a pathogenic bacteria, and the neutrophils serve to diminish local damage in the setting of a cerebritis.

Acute Disease↗

PubMed1998

Mammalian glycophosphatidylinositol anchor transfer to proteins and posttransfer deacylation.

The glycophosphatidylinositol (GPI) anchors of proteins expressed on human erythrocytes and nucleated cells differ with respect to acylation of an inositol hydroxyl group, a structural feature that modulates their cleavability by PI-specific phospholipase C (PI-PLC). To determine how this GPI anchor modification is regulated, the precursor and protein-associated GPIs in two K562 cell transfectants (ATCC and .48) exhibiting alternatively PI-PLC-sensitive and resistant surface proteins were analyzed and the temporal relationship between GPI protein transfer and acquisition of PI-PLC sensitivity was determined. Nondenaturing PAGE analyses demonstrated that, whereas in .48 transfectants the GPI anchors in decay accelerating factor (DAF) and placental alkaline phosphatase (PLAP) were >95% acylated, in ATCC transfectants, they were 60 and 33% unsubstituted, respectively. In contrast, TLC analyses revealed that putative GPI donors in the two lines were identical and were >/=95% acylated. Studies of de novo DAF biosynthesis in HeLa cells bearing proteins with >90% unacylated anchors showed that within 5 min at 37 degreesC (or at 18 degreesC, which does not permit endoplasmic reticilum exit), >50% of the anchor in nascent 44-kDa proDAF protein exhibited PI-PLC sensitivity. In vitro analyses of the microsomal processing of miniPLAP, a truncated PLAP reporter protein, demonstrated that the anchor donor initially transferred to prominiPLAP was acylated and then progressively was deacylated. These findings indicate that (i) the anchor moiety that initially transfers to nascent proteins is acylated, (ii) inositol acylation in mature surface proteins is regulated via posttransfer deacylation, which in general is cell-specific but also can be protein-dependent, and (iii) deacylation occurs in the endoplasmic reticulum immediately after GPI transfer.

Acylation↗

PubMed1998

Crossed reduction of human motor cortex excitability by 1-Hz transcranial magnetic stimulation.

Electrophysiological studies have shown that 1-Hz repetitive transcranial magnetic stimulation (rTMS) of the primary motor area (M1) can produce a local decrease in excitability. Functional imaging data suggest that this change may be bilateral. In normal subjects, we measured motor evoked potential (MEP) amplitude at a series of stimulation intensities in the contralateral M1 before and after 15 min of active or sham rTMS at just above the MEP threshold. The slope of the curve relating MEP amplitude and stimulation intensity was decreased in the unstimulated hemisphere by active but not sham rTMS. This demonstrates that rTMS can condition cortical excitability at a distance of one or more synapses and suggest that decreased excitability to TMS is a correlate of decreased blood flow and metabolism.

Adult↗

PubMed1998

Inhibitory influence of the ipsilateral motor cortex on responses to stimulation of the human cortex and pyramidal tract.

1. The ability of the primary motor cortex (M1) to modulate motor responses in ipsilateral hand muscles seems to be important for normal motor control and potentially also for recovery after brain lesions. It is not clear which pathways mediate this ipsilateral modulation. Transcallosal connections have been proposed, but are known to be sparse between cortical hand motor representations in primates. The present study was performed to determine whether descending ipsilateral modulation of motor responses might also be mediated below the cortical level in humans. 2. A paired-pulse protocol was used, in which motor-evoked potentials (MEPs) were produced by cortical transcranial magnetic stimulation (cTMS) or by electrical stimulation of the pyramidal tract at the level of the pyramidal decussation (pdTES), in both preactivated and relaxed hand muscles. Paired stimuli were applied at various interstimulus intervals (ISIs) between 2 and 100 ms. The conditioning stimulus (CS) was always magnetic, and delivered to the M1 ipsilateral to the target hand, prior to the test stimulus (TS). The magnetic TS was delivered to the M1 contralateral to the target hand; the electrical TS was applied through electrodes placed over the mastoid process bilaterally. Further experiments included cortical electrical stimulation and H-reflexes. The MEP amplitudes were averaged separately for each ISI and the control condition (no CS), and expressed as a percentage of the unconditioned response. 3. Conditioning stimulation of the ipsilateral M1 resulted in significant inhibition of magnetically evoked MEPs, and also of MEPs produced by pdTES. Inhibition occurred at ISIs between 6 and 50 ms, and was observed in preactivated and relaxed muscles. Higher CS intensities caused greater inhibition of both cTMS- and pdTES-evoked MEPs. 4. While the conditioning effects on magnetically evoked muscle responses could be explained by a transcallosal mechanism, the effects on pdTES-evoked MEPs cannot, because they are elicited subcortically and are therefore not susceptible to inhibitory mechanisms transmitted at the cortico-cortical level. 5. In conclusion, the present results provide novel evidence that the inhibitory influence of the human M1 on ipsilateral hand muscles is to a significant extent mediated below the cortical level, and not only through cortico-cortical transcallosal connections. They point to a concept of inhibitory interaction between the two primary motor cortices that is relayed at multiple levels along the neuroaxis, thus perhaps providing a structurally redundant system which may become important in case of lesions.

Adult↗

PubMed1998

Mechanisms of cortical reorganization in lower-limb amputees.

The human motor system undergoes reorganization after amputation, but the site of motor reorganization and the mechanisms involved are unknown. We studied the site and mechanisms of motor reorganization in 16 subjects with traumatic lower-limb amputation. Stimulation at different levels in the CNS was used to determine the site of reorganization. The mechanisms involved were evaluated by measuring the thresholds for transcranial magnetic stimulation (TMS) and by testing intracortical inhibition and facilitation. With TMS, the threshold for muscle activation on the amputated side was lower than that of the intact side, but with transcranial electrical stimulation there was no difference in motor threshold between the two sides. TMS at the maximal output of the stimulator activated a higher percentage of the motor neuron pool (%MNP) on the amputated side than on the intact side. The %MNP activated by spinal electrical stimulation was similar on the two sides. Paired TMS study showed significantly less intracortical inhibition on the amputated side. Our findings suggest that motor reorganization after lower-limb amputation occurs predominately at the cortical level. The mechanisms involved are likely to include reduction of GABAergic inhibition.

Adult↗

PubMed1998

Antibacterial agents that inhibit two-component signal transduction systems.

A class of antibacterials has been discovered that inhibits the growth of Gram-positive pathogenic bacteria. RWJ-49815, a representative of a family of hydrophobic tyramines, in addition to being a potent bactericidal Gram-positive antibacterial, inhibits the autophosphorylation of kinase A of the KinA::Spo0F two-component signal transduction system in vitro. Analogs of RWJ-49815 vary greatly in their ability to inhibit growth of bacteria and this ability correlates directly with their activity as kinase A inhibitors. Compared with the potent quinolone, ciprofloxacin, RWJ-49815 exhibits reduced resistance emergence in a laboratory passage experiment. Inhibition of the histidine protein kinase::response regulator two-component signal transduction pathways may present an opportunity to depress chromosomal resistance emergence by targeting multiple proteins with a single inhibitor in a single bacterium. Such inhibitors may represent a class of antibacterials that potentially may represent a breakthrough in antibacterial therapy.

Anti-Bacterial Agents↗

PubMed1998

Regulated expression of mature human insulin in the liver of transgenic mice.

Transgenic mice expressing either human proinsulin cDNA or mutated proinsulin cDNA in the liver were created. The human proinsulin cDNA was mutated to generate a protein cleavable by the ubiquitous prohormone convertase furin, thus leading to mature insulin peptide. All transgenic lines expressed human C-peptide in the blood, whose level varied according to nutritional conditions. High performance liquid chromatography fractionation of mouse serum revealed that mutant proinsulin was effectively processed into mature insulin in vivo. This transgenic mouse model provides a useful tool for further prospects of gene therapy of insulin-dependent diabetes mellitus.

Animals↗

PubMed1998

Molecular cloning of human homolog of yeast GAA1 which is required for attachment of glycosylphosphatidylinositols to proteins.

Anchoring proteins to cell surface membranes by glycosylphosphatidylinositols (GPIs) is important. We have isolated a component of the putative transamidase machinery, hGaa1p (human GPI anchor attachment protein). hGAA1 cDNA is approximately 2 kb in length and codes 621 amino acids. The amino acid sequence of hGaa1p is 25%, identical and 57% homologous to that of yeast Gaa1p. Moreover, Kite-Dolittle hydrophobicity plots of both proteins show marked similarity. hGAA1 gene is expressed ubiquitously and mRNA levels are higher in the undifferentiated state. Overexpression of antisense hGAA1 in human K562 cells significantly reduced the production of a reporter GPI-anchored protein.

Amino Acid Sequence↗

PubMed1998

Time course of corticospinal excitability in reaction time and self-paced movements.

We used transcranial magnetic stimulation (TMS) to study the time course of corticospinal excitability before and after brisk thumb abduction movements, either in a simple reaction time (RT) paradigm or self-paced. Premovement increase in corticospinal excitability began about 20 msec earlier for self-paced compared with simple RT movements. For both simple RT and self-paced movements after electromyographic (EMG) offset, there was a first period of increased excitability from 0 to 100 msec, followed by a second period from 100 to 160 msec. Corticospinal excitability was decreased from about 500 to 1,000 msec after EMG offset for both types of movements. Our results show that motor preparation that begins 1.5 to 2 seconds before self-paced movement is not associated with increased corticospinal excitability. The first phase of increased corticospinal excitability after EMG offset may be due to activity of motor cortex neuron subthreshold for activating spinal motor neurons, and the second phase may reflect a subthreshold second agonist burst. The period of decreased corticospinal excitability after movement corresponds to the onset of event-related synchronization (ERS) of electroencephalographic signals in the 20-Hz band, and supports the hypothesis that ERS may be related to an inactive, idling state of the motor cortex.

Adolescent↗

PubMed1998

Cytoskeletal changes in platelets induced by thrombin and phorbol myristate acetate (PMA).

Changes in shape, and aggregation that accompanies platelet activation, are dependent on the assembly and reorganization of the cytoskeleton. To assess the changes in cytoskeleton induced by thrombin and PMA, suspensions of aspirin-treated,32P-prelabeled, washed pig platelets in Hepes buffer containing ADP scavengers were activated with thrombin, and with PMA, an activator of protein kinase C. The cytoskeletal fraction was prepared by adding Triton extraction buffer. The Triton-insoluble (cytoskeletal) fraction isolated by centrifugation was analysed by SDS-PAGE and autoradiography. Incorporation of actin into the Triton-insoluble fraction was used to quantify the formation of F-actin. Thrombin-stimulated platelet cytoskeletal composition was different from PMA-stimulated cytoskeletal composition. Thrombin-stimulated platelets contained not only the three major proteins: actin (43 kDa), myosin (200 kDa) and an actin-binding protein (250 kDa), but three additional proteins of Mr56 kDa, 80 kDa and 85 kDa in the cytoskeleton, which were induced in by thrombin dose-response relationship. In contrast, PMA-stimulated platelets only induced actin assembly, and the 56 kDa, 80 kDa and 85 kDa proteins were not found in the cytoskeletal fraction. Exposure of platelets to thrombin or PMA induced phosphorylation of pleckstrin parallel to actin assembly. Staurosporine, an inhibitor of protein kinase C, inhibited actin assembly and platelet aggregation induced by thrombin or PMA, but did not inhibit the incorporation of 56 kDa, 80 kDa and 85 kDa into the cytoskeletal fraction induced by thrombin. These three extra proteins seem to be unrelated to the induction of protein kinase C. We conclude that actin polymerization and platelet aggregation were induced by a mechanism dependent on protein kinase C, and suggest that thrombin-activated platelets aggregation could involve additional cytoskeletal components (56 kDa, 80 kDa, 85 kDa) of the cytoskeleton, which made stronger actin polymerization and platelet aggregation more.

Actins↗

PubMed1998

Isradipine prevents global and regional cocaine-induced changes in brain blood flow: a preliminary study.

The L-type calcium channel antagonist, isradipine, reduces brain ischemia in animal models of ischemic stroke. These effects of isradipine appear more pronounced in dopamine (DA) rich brain regions. These same DA-rich brain regions have also been shown to be the areas most affected by cocaine-induced ischemic changes. Using a novel quantified approach to single photon emission computerized tomography, we demonstrated that isradipine pre-treatment prevented cocaine-induced ischemic changes, especially in these DA-rich brain regions. This is the first demonstration that any medication, including isradipine, can prevent the ischemic effects of cocaine on brain blood flow. Isradipine may, therefore, be a useful therapeutic agent for the prevention of brain ischemia in cocaine addicts.

Adult↗

PubMed1998

Plant NADP-dependent isocitrate dehydrogenases are predominantly localized in the cytosol.

The isoenzyme patterns of NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) have been investigated in 15 species of higher plants using dietylaminoethyl ion-exchange chromatography and immunological techniques. The obtained results unambiguously demonstrate that the cytosolic enzyme is the predominant form in leaf extracts of all the surveyed plant species. The chloroplastic isoenzyme, previously reported in pea (Pisum sativum L.) leaves (R.D. Chen et al., 1989, Planta 178; 157-163), is a minor form in ferns and dicotyledonous angiosperms and is undetectable in gymnosperms and monocotyledonous angiosperms. Comparison of immunological relatedness suggests that the proteins of cytosolic isoenzymes have been highly conserved in the course of plant evolution. The data support the previously proposed idea that the cytosol is the major site for alpha-ketoglutarate production to be used for nitrogen assimilation.

Cytosol↗

PubMed1998

A relaxin-mediated pathway to preterm premature rupture of the fetal membranes that is independent of infection.

OBJECTIVE: This study was designed to show whether the overexpression of relaxin in the decidua of patients with preterm premature rupture of the membranes is independent of or a consequence of chorioamnionitis. STUDY DESIGN: Two experiments were conducted. In the first experiment fetal membranes and decidua were collected from patients with preterm premature rupture of the membranes (n = 17) or preterm labor (n = 17) and were divided according to their degree of histologic infection. Messenger ribonucleic acid was isolated from the tissues and quantitative, sequential Northern analyses were carried out for the expression of human relaxin, interleukin-1beta, interleukin-6, and interleukin-8. The second experiment was aimed at increasing the numbers of messenger ribonucleic acid preparations in the two extreme categories, uninfected and severely infected tissues, with preterm premature rupture of the membranes and preterm labor. Some samples of messenger ribonucleic acid from the first experiment were rerun with the Northern analyses in the second experiment. These repeat samples showed no statistical differences in the results run at different times. Therefore the data from the respective groups of patients in both experiments were pooled for statistical analysis. RESULTS: In both the first experiment and in the pooled data of the two experiments the expression of the relaxin genes was significantly greater (P < .005) in the tissues from patients with preterm premature rupture of the membranes compared with those with preterm labor, in the absence of infection. No effect of the level of infection on the expression of relaxin was noted. In contrast, interleukin-6 gene expression was significantly increased (P < .05) in severely infected tissues, which was independent of whether the delivery was from preterm premature rupture of the membranes or preterm labor. The expression of the interleukin-1beta and interleukin-8 genes were only marginally increased even in severe infection. Marked patient variability in expression of the interleukin genes, especially in severe infection, was noted. CONCLUSION: A relaxin-mediated pathway that leads to preterm premature rupture of the membranes may exist independent of infection.

Adult↗

PubMed1998

Fluoride profiles of perikymata in enamel surfaces of human premolars.

Twenty-five premolars (from Nagoya, Japan < 0.1 parts/10(-6)F in drinking water) were sampled to determine the fluoride content in imbrication lines of Retzius between the grooves and ridges of perikymata on the enamel surface. Eight small windows were formed on each surface in groove and ridge regions using an etched microsampling technique. By using a regression curve, y = ax(-b), fluoride concentrations were compared at depths of 1, 3, 5, 10, 20, 30 and 50 microm in the perikymata regions. Fluoride concentrations increased gradually from the age of 10 to 12 years on the premolar surfaces, but were significantly higher in 12-year-olds or older (erupted teeth) than in 10-year-olds or younger (unerupted teeth). No obvious difference in fluoride concentrations was found between males and females. In the outermost enamel ( < 5 microm depth), fluoride concentrations were significantly higher in the grooves than the ridges of the perikymata. Comparison of the exponential regression coefficients (-b) of the fluoride profiles showed a significant difference between the grooves and ridges. It was concluded that fluoride concentrations and profiles were higher in grooves than in ridges of perikymata, probably because they are naturally porous and are stagnation areas attracting dental plaque.

Acid Etching, Dental↗

PubMed1998

Clinical significance of fever after percutaneous nephrolithotomy.

OBJECTIVES: In the immediate postoperative period after percutaneous stone removal, body temperature elevations are common. Pyrexia after a percutaneous nephrolithotomy (PCNL) generates concern because of the possibility of urinary extravasation and bacteremia. We reviewed our experience with PCNL to determine the clinical significance of a postoperative fever before discharge from the hospital. METHODS: Between July 1994 and December 1996, 63 patients underwent 69 PCNLs. Each had documented negative urine cultures preoperatively and received prophylactic antibiotics at the time of surgery. For each case, clinical and operative charts were reviewed to determine stone composition, fever during hospital stay, postoperative bacteriologic cultures, postoperative white blood cell count (WBC), and clinical course. RESULTS: Complete data were available for 66 procedures. Eight patients (12%) had at least one body temperature reading between 38.0 and 38.5 degrees C. Eleven patients (16.7%) had at least one temperature greater than 38.5 degrees C. Each patient with a temperature greater than 38.5 degrees C was hemodynamically stable with negative blood and urine cultures. No patient with a fever between 38.0 and 38.5 degrees C was cultured. Stone analysis did not demonstrate any association between postoperative fever and stone composition (including 22 struvite stones). Postoperative WBC also did not predict pyrexia. Fever alone did not prolong hospital stay. CONCLUSIONS: In patients with negative urine cultures who are prophylaxed with immediate preoperative antibiotics and maintained on postoperative antibiotics, pyrexia after PCNL does not require an immediate bacteriologic evaluation in those who are hemodynamically stable.

Female↗

PubMed1998

Demonstration of dose-dependent global and regional cocaine-induced reductions in brain blood flow using a novel approach to quantitative single photon emission computerized tomography.

Ischemic stroke is a common cause of morbidity and mortality in cocaine addicts. Because the previous semiquantitative single photon emission computerized tomography (SPECT) method for measuring brain blood flow does not quantify blood flow, the magnitude and specificity of cocaine's effects during drug taking has not been well established. Here, using a novel quantitative approach to SPECT, we established that intravenous cocaine administration to nine recently abstinent cocaine-dependent subjects was associated with significant decreases in global and regional brain blood flow to dopamine-rich areas such as the prefrontal, frontal temporal, and subcortical gray matter. Establishing the utility of this relatively new quantitative SPECT technique provides an important tool for the management of vascular disorders of the brain. Additionally, identifying the site-specific effects of cocaine provides targets for the development of putative therapeutic medications to attenuate or minimize ischemic stroke in cocaine addicts.

Adolescent↗

PubMed1998

Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development.

Corticotropin releasing factor (CRF) is a major integrator of adaptive responses to stress. Two biochemically and pharmacologically distinct CRF receptor subtypes (CRFR1 and CRFR2) have been described. We have generated mice null for the CRFR1 gene to elucidate the specific developmental and physiological roles of CRF receptor mediated pathways. Behavioral analyses revealed that mice lacking CRFR1 displayed markedly reduced anxiety. Mutant mice also failed to exhibit the characteristic hormonal response to stress due to a disruption of the hypothalamic-pituitary-adrenal (HPA) axis. Homozygous mutant mice derived from crossing heterozygotes displayed low plasma corticosterone concentrations resulting from a marked agenesis of the zona fasciculata region of the adrenal gland. The offspring from homozygote crosses died within 48 hr after birth due to a pronounced lung dysplasia. The adrenal agenesis in mutant animals was attributed to insufficient adrenocorticotropic hormone (ACTH) production during the neonatal period and was rescued by ACTH replacement. These results suggest that CRFR1 plays an important role both in the development of a functional HPA axis and in mediating behavioral changes associated with anxiety.

Adaptation, Physiological↗